bluuebunny/crossref_metadata_2025 huggingface dataset:
Prosections in Anatomy Education: Preparation, Use, and Educator Perceptions
Authors: Curran Shannon , Schaefer Audra
Published: April 2020

In response to decreasing anatomy lab contact hours, some anatomy courses are using prosections in order to maintain the benefits of utilizing human cadaveric donors while reducing student lab time. This change has spurred an ongoing debate amongst anatomy educators regarding the best method of anatomy instruction: student dissection or use of prosection? A missing piece in this debate is information on what methods of preparation and use of prosections will provide the best student outcomes. There are a variety of ways to prepare prosections for educational use that potentially influence their effectiveness as teaching tools. While there is plenty of literature comparing dissection and prosection, there are no studies examining how prosections are prepared and used in anatomy instruction. In this IRB approved study, an anonymous survey will be distributed online to anatomy educators to determine how human cadaveric prosections are prepared and utilized in courses they teach at their institution. Anatomy educators from all levels of higher education and all program types will be invited to provide input. The aims of this study are to determine: 1) anatomy educator’s views on the use of prosection in anatomy education, focusing on how they define prosection and what they think the strengths and weaknesses of prosection use in anatomy education are, 2) the types of institutions and course levels using prosections for anatomy education, 3) how prosections are used as either a supplemental tool or a primary teaching tool, 4) how prosections are prepared for different uses, 5) who prepares prosections at different institutions, and 6) how it is decided that prosections will be used compared to other resources in anatomy education. Descriptive statistics will be performed on quantitative data collected from multiple choice questions and free response survey items will undergo thematic analyses to determine themes amongst responses. Finding out how educators are preparing and utilizing prosections is the first step towards determining how best to maximize the effectiveness of prosection use. The results of this study will lay the foundation and provide background information for further prosection use and preparation studies.
Novel insights into the fucose metabolism – challenging the old dogma
Authors: Sosicka Paulina , Ng Bobby G. , Wong Maurice , Xia Zhi-Jie , Scott David , Lebrilla Carlito B. , Freeze Hudson H.
Published: April 2020

GDP‐Fucose used for glycan fucosylation is synthesized de novo from either glucose (Glc) or mannose (Man) or utilized directly from salvaged/exogenous fucose (Fuc). Meticulous radioisotope dilution studies performed in the 1970’s only in HeLa cells using 0.3 μM Fuc showed that the de novo pathway provides >90% of GDP‐Fuc and salvage <10%.

Employing different Fuc‐specific lectins, we showed that at low concentrations (<20 μM), the entry of Fuc into cells relies on transport via GLUT1 (SLC2A1), but above this, most Fuc enters via amiloride‐sensitive macropinocytosis. Inhibitors of both clathrin‐ and caveolin‐dependent endocytosis had no effect on uptake. These findings were further confirmed using 3H Fuc in four cell lines.

We used differentially labelled 13C Glc, Man and Fuc to study contributions of each pathway to Fuc in N‐glycans in six cell lines. Very low concentrations (0.5–30 μM) of exogenous Fuc progressively inhibit the de novo pathway, and completely by ~50 μM. Surprisingly, GDP‐Fuc derived from Glc is much more sensitive to this inhibition than that derived from Man, even though both contribute to GDP‐Man synthesis. When cultured in 30–50 μM Fuc, cells not only inhibit the de novo pathway, but also prefer exogenous over salvaged Fuc. Cells seem to distinguish multiple pools of GDP‐Fuc based on their origin.

Using LC‐MS/MS and eight different cell lines, we studied the ongoing biosynthesis of >150 fucosylated N‐glycans containing 1–4 Fuc residues by comparing the proportion of 13C Fuc incorporation at various concentrations for 24h. Over the labeling period, cells had to choose between GDP‐Fuc synthesized de novo or from exogenous fucose. N‐glycans with a single Fuc show a proportional increase in 13C‐labeled species with increasing concentration of label. To our surprise, glycans with 2, 3, and 4 Fuc each required progressively higher exogenous 13C Fuc to become fully labeled.

Based on LC‐MS/MS and lectin staining, 1–6αFuc labels more efficiently with exogenous Fuc than 1–2α, 1–3α and 1–4αFuc, suggesting differential access to exogenous sugar that depends partially on Fuc linkage. Km differences between fucosyltransferases cannot explain preferential use of GDP‐Fuc synthesized from exogenous monosaccharide over de novo produced substrate. Rather, it suggests the existence of separate, ill‐defined pools of GDP‐Fuc. Altogether it raises the question whether salvaged fucose equivalent to exogenous Fuc? Their behavior clearly differs from de novo synthesized GDP‐Fuc.

Support or Funding Information

The Rocket Fund and R01DK099551.
“A Whole New Perspective on How the Body Fits Together” – An Evaluation of a Cadaver Lab Experience for High School and Undergraduate Students
Authors: Cale Andrew , Byram Jessica , Schmalz Naomi
Published: April 2020

Background

Since 2014, the Center for Anatomy and Physiology Education at Indiana University School of Medicine – Indianapolis has hosted interactive human cadaver lab tours for local high school and undergraduate students. During these graduate student‐led tours, touring students observe human anatomy on prosections and with isolated organs. Similar educational outreach initiatives have been shown to benefit adolescents by improving health literacy and encouraging interests in healthcare careers.

Aim

To evaluate students’ perceptions of the anatomy lab tours and their impact on students’ interests in healthcare careers.

Methods

Between April and December 2018, touring students (n=261) were invited to complete pre‐ and post‐tour surveys evaluating their past experiences with cadaver labs, tour expectations, career interests, and additional thoughts and feelings regarding the tour. Pre‐surveys were paper‐based and completed in‐person, while post‐surveys were administered online via REDCap and completed within one week of the tour. Responses were aggregated into pre‐ and post‐tour groups and analyzed using thematic analysis. This study was granted exempt status from the IRB at Indiana University (#1802755251).

Results

Of the 261 students who completed pre‐tour surveys, 204 (78%) completed the post‐tour survey. Before the tour, students anticipated learning about human anatomy (52%) and expected to only be able to see (and not feel) a cadaver (66%). Most students expressed excitement (80%) or nervousness (45%). A few students viewed the lab tour as an opportunity to test if they could see themselves in a healthcare career (4%). After the tour, most students indicated that the tour either met (77%) or exceeded (22%) their expectations. Students found the lab tour to be educational and interesting. They were surprised by the opportunity to interact with the cadaver (23%) and by the pre‐dissected appearance of the cadaver (26%). Numerous students (41%) also expressed an increased interest in healthcare careers after the lab tour.

Conclusion

Overall, students perceived the lab tours as engaging, educational experiences that not only improved their anatomical knowledge, but also reinforced or increased their interest in healthcare careers. Academic institutions interested in positively impacting their local students can implement a cadaver lab tour program by sharing access to their in‐house human cadaver lab and recruiting instructors to share their time and anatomy expertise.
The efficacy of Augmented Reality in improving the learning outcome
Authors: Bakhtiari Shahram
Published: April 2020

Augmented Reality is gaining traction as an alternative and effective learning tool in studying human anatomy in colleges and universities. This technology provides an immersive approach to learn anatomical structure using HoloLens with an extensive range of maneuverability of Hologram, dissection mode, information boxes and most amazingly walking into the Hologram and visualizing the human body inside out.

Human anatomy, as one of the introductory courses in health sciences is usually found challenging for the students and the capability of any learning tools to provide simplification and three‐dimensional visualization, in addition to eliciting excitement with advanced technology will be promising to improve the students’ learning experience.

The inclusion of the Augmented Reality into the human anatomy course was able to improve the students’ final exam grade 18.9% and reduce the failure rate by 10%. The student’s satisfaction survey also showed 87.6% of the students believed that the Augmented Reality helped them to reach the objectives of the anatomy course.

Support or Funding Information

West Coast University
The DSP Domain of Pseudophosphatase MK‐STYX Decreases Stress Granules
Authors: Jiang Fei
Published: April 2020

MK‐STYX [MAPK (mitogen activated protein kinase) phosphoserine/threonine/tyrosine binding protein] is a pseudophosphatase. Due to point mutations in the HC(X5)R catalytic active site signature motif, MK‐STYX (FSTQGISR) can only bind, but cannot dephosphorylate phosphorylated residues. This interaction of MK‐STYX allows it to play important roles in various cellular pathways including the stress response pathway. MK‐STYX decreases stress granules (SG) independently of serine 149 phosphorylation of G3BP‐1 (stress granule nucleator). Stress granules are cytoplasmic aggregates of untranslated mRNA and proteins formed in response to stressors. The aim of this study is to determine which domain of MK‐STYX decreases stress granules. MK‐STYX has two domains: a dual specificity phosphatase domain (DSP) and a rhodanese/cdc 25 phosphatase homology domain. In our study, HEK‐293 cells are cotransfected with G3BP‐GFP and mCherry‐DSP, mCherry‐CH2, or mChrerry. Cells expressing G3BP‐GFP and mCherry‐DSP exhibit fewer SG than the control (G3BP‐GFP and mCherry). Whereas, cells cotransfected with G3BP‐GFP and mCherry‐CH2 have similar levels of SG compared to the control, indicating that the DSP domain of MK‐STYX is responsible for decreasing the number of stress granules. Since the “active site” lies within the DSP domain, the study suggests that MK‐STYX decreases SG by binding, and perhaps blocking dephosphorylation of certain proteins. Future studies will focus on characterizing size and exact number of these SGs, as well as the interactors of MK‐STYX‐DSP.
Agonism of adiponectin Receptor Prevents 7‐ketocholesterol‐induced Endothelial Inflammation through Suppressing Endoplasmic Reticulum and Oxidative Stresses
Authors: Zhang Yang , Chen Jiajie , Boini Krishna , Li Xiang
Published: April 2020

Endoplasmic reticulum (ER) stress is involved in endothelial inflammation and injury by various cardiovascular risk factors including oxysterols. AdipoRon is a selective small‐molecule agonist of adiponectin receptor, which has been shown to mimics many beneficial effects of adiponectin on the cardiovascular system including endothelial cells. However, the molecular mechanisms that mediate adipoRon‐induced endothelial protection are not well understood. Here, we studied whether adipoRon treatment interferes with the induction of ER stress to protect the endothelial cells from oxysterol‐induced inflammation and injury in vitro and to inhibit vascular inflammation from acute hypercholesterolemia in vivo. In cultured murine endothelial cells, adipoRon prevented 7‐ketocholesterol‐induced increases in endothelial cell death, inflammatory gene expression, monocyte adhesion, and Nlrp3 inflammasome activation. In parallel, adipoRon inhibited 7‐ketocholesterol‐induced ER stress and reactive oxygen species (ROS) production. In mice with acute hypercholesterolemia, adipoRon pre‐treatment could inhibit ROS production and ER stress in the coronary arterial wall without alterations in the plasma cholesterol concentration. Moreover, adipoRon reduced expression of ICAM1 in the coronary arterial intima and decreased numbers of CD45+ immune cells that adhere to or accumulate around the coronary arteries. Together, our results indicate that the cardiovascular protective action of adipoRon is associated with its inhibitory effects on ROS production and ER stress and consequent inflammatory responses in endothelial cells.

Support or Funding Information

National Institutes of Health (HL122769 and HL122937)
The In’s and Out’s of Science Outreach: Assessment of an Engaging New Program
Authors: Metz Cynthia J. , Metz Michael J.
Published: April 2020

Many professors are interested in providing science outreach to local K‐12 students, but may not have the time or resources needed to create an engaging presentation. The American Physiological Society supports outreach initiatives through programs such as the Physiology Understanding (PhUn) Week. Building upon these activities, it was desired to create and assess an immersive and engaging outreach program in a disadvantaged K‐12 student population. Three distinct modules were created on cardiovascular physiology, respiratory physiology, and oral health. All resources for these modules underwent peer‐review and publication, allowing other professors to easily execute their own outreach programming. The outreach modules were presented to 288 students in 3rd, 5th, 6th, and 7th grade classes. Implementation of the modules resulted in significant increases in student content knowledge, ranging from 32–57% improvement (P<0.001, dependent t‐test) with an average increase of 46%. K‐12 science teachers reported that the program was at an appropriate educational level, increased students’ enthusiasm for science, and increased students’ exposure to science careers. Additionally, the presenters of the outreach program were perceived to be enthusiastic, knowledgeable, and proficient at interacting with the students. On open‐response survey items, the science teachers indicated a high level of satisfaction with the program and an enthusiasm for continued collaborations. These results indicate the importance of organized and interactive science activities for the success of a new outreach program.

Support or Funding Information

This work was supported in part by a basic grant from the University of Louisville School of Medicine and a support grant from the Dean’s Office of the University of Louisville School of Dentistry.
The protein kinase TNIK: a novel druggable target in Lung Squamous Cell Carcinoma with 3q amplification.
Authors: Ayuso Pedro Torres , An Elvira , Ritt Daniel , Nyswaner Katherine , Specht Suzanne , Liang Roger , Das Sudipto , Andresson Thorkell , Turk Benjamin , Morrison Deborah , Brognard John
Published: April 2020

Lung squamous cell carcinoma (LSCC) is the second most common type of lung cancer. No precision therapies have been approved for the treatment of LSCC and the genetic drivers of LSCC are unknown in approximately 60% of the cases. As a result, treatment of LSCC is still limited to chemotherapy and/or radiotherapy. Analysis of LSCC samples from the TCGA reveals that nearly 50% of LSCC patients harbor distal amplification of the 3q chromosome that includes the resident protein kinase gene TNIK. Recent studies have pinpointed TNIK as a potential oncogenic driver in cancer cells with distal 3q amplification (Cancer Discov. 2013; 3:1044). However, the therapeutic potential of TNIK in LSCC remains unexplored.

To determine if amplified TNIK is a genetic driver in LSCC, we analyzed TNIK expression in a panel of LSCC cell lines. We show that TNIK is overexpressed in LSCC cell lines in comparison with primary lung cells from healthy donors, consistent with observations in patient tumors based on analysis of TCGA data. Furthermore, we demonstrate that TNIK genetic depletion or pharmacological inhibition significantly reduces survival of LSCC cells with 3q amplification in vitro and in in vivo LSCC cell‐line derived mouse xenografts. Importantly, we have generated LSCC patient‐derived xenograft (PDX) mouse models to test TNIK inhibitors and have observed that inhibition of TNIK catalytic activity suppresses tumor growth in LSCC PDX mouse models.

To elucidate the molecular mechanisms underpinning TNIK‐driven LSCC cell viability we have used a combination of reverse‐phase protein arrays, motif‐driven screens for proteins with TNIK consensus phosphorylation sites, co‐expression experiments, peptide mapping and mass spectrometry. Through these approaches, we have identified the tumor suppressor MERLIN as a novel TNIK substrate and determined that TNIK phosphorylates MERLIN at multiple sites.

In conclusion, we have pinpointed the protein kinase TNIK as a promising therapeutic target in LSCC that maintains survival of LSCC cells through modulation of novel a TNIK‐MERLIN signaling pathway.

Support or Funding Information

PT‐A has been funded by Foundation Ramon Areces. This work has been supported by grant ZIABC011691 to J.B.
Muscle Fibers Utilize its Glucose Uptake Machinery to Reseal Sarcolemma Disruptions & Stimulating Affiliated Rab G Proteins Improves Repair Capacity in Muscle Dystrophic Mice
Authors: Kwiatkowski Thomas A , Rose Aubrey , Jung Rachel , McElhanon Kevin , Paleo Brian , Beck Eric X , Weisleder Noah
Published: April 2020

The glucose uptake machinery in cells and muscle fibers is a robust and highly regulated system used to import extracellular glucose for intracellular metabolic processing. The majority of glucose uptake in skeletal muscle is done through stimulated exocytosis and fusion of vesicles to the plasma membrane to enrich the cellular exterior with glucose transporter protein GLUT4. In our investigation, we found evidence that GLUT4 containing vesicles (GSV) and the glucose uptake regulators AS160, rab8A, and rab10 serve an additional role in the plasma membrane repair response. Through the use of mutant plasmids and live cell imaging, we observed that GSVs, as well as affiliated rabs, translocate to injury sites in C2C12 myoblasts and isolated mouse muscle fibers under different modes of damage. We observed that AS160 phosphorylation from the phosphoinositide‐3 kinase (PI3K)/Akt1 signaling axis regulates membrane repair in cultured muscle cells. In addition, we screened GSV associating rab G proteins and saw that knocking down rab8A and rab10 expression reduced the repair capacity of the cells. Importantly, through the electroporation of overexpression or constitutively active rab10 mutant constructs in skeletal muscle of Duchenne muscular dystrophy mdx mice, there was a significant improvement in membrane resealing in vivo. Overall, our findings suggest that the glucose uptake vesicles commonly thought of as important for metabolic homeostasis, serve an additional function in repairing membrane wounds in some cell lines and mouse muscle fibers. Targeting glucose uptake regulators could lead to the development of new therapies for muscle disease and injury.
Sympathetic Vascular Transduction is Elevated in Hypertensive Premenopausal and Postmenopausal Women
Authors: Adams Zoe H. , Kobetic Matthew D. , Ratcliffe Laura E. , Hope Katrina A. , Paton Julian F. R. , Nightingale Angus K. , Hart Emma C.
Published: April 2020

The transduction of muscle sympathetic nerve activity (MSNA) to vasoconstrictor tone is lower in healthy young women versus both healthy young men and healthy postmenopausal women, and may account for the lower risk of hypertension in young women. However, hypertension can occur before menopause in some women but whether sympathetic transduction is increased in this group is unknown. We aimed to determine whether sympathetic transduction is different in hypertensive (HTN) pre‐ and post‐menopausal women relative to normotensive (NTN) controls. MSNA (peroneal microneurography) and continuous blood pressure (Finometer Pro) were measured in HTN (n=5) and NTN (n=5) premenopausal and HTN (n=4) and NTN (n=6) postmenopausal women. HTN and NTN groups were matched for age (premenopausal; 46±2 vs. 46±5 years, p=0.97, postmenopausal; 59±4 vs. 62 ±2 years; p=0.17). Sympathetic transduction was measured by associating beat‐to‐beat diastolic blood pressure (DBP) with MSNA burst area between the 6th and 8th previous cardiac cycles to account for transduction delay. Transduction was expressed as the slope of the linear regression between burst area and mean DBP, plotted as 1 %.s bins. HTN and NTN were compared within pre‐ or postmenopausal groups by unpaired Student’s T‐test or Mann‐Whitney test, where data were not normally distributed. Data are presented as mean ± SD. Both sympathetic transduction and MSNA were greater in HTN versus NTN premenopausal women (0.16±0.11 vs. 0.03±0.02 mmHg/%.s, p=0.03; 57±17 vs. 32±7 bursts/100 heartbeats, p=0.0185). Sympathetic transduction was also greater in HTN vs. NTN postmenopausal women (0.19±0.08 vs. 0.07±0.02 mmHg/%.s, p=0.02), although MSNA was similar (53±8 vs. 60±15 bursts/100 heartbeats, p=0.4255). Both HTN groups had increased clinic systolic BP and DBP versus the respective NTN group (premenopausal women 153±11/97±6 vs. 119±9/73±8 mmHg, p=0.0007 and p=0.0079; postmenopausal women 155±8/92±15 vs. 123±8/70±7 mmHg, p=0.0095 and p=0.0132 respectively). These results indicate that sympathetic transduction is elevated in both premenopausal and postmenopausal women with HTN vs. NTN controls. As such, increased sympathetic transduction may help to explain the development of hypertension in premenopausal women. These data will assist with more precise management of hypertension in pre‐ and postmenopausal women.

Support or Funding Information

Zoe Adams is funded by a British Heart Foundation Non‐Clinical PhD Studentship (FS/17/38/32935).
Evaluation of Potential Human Sodium Taurocholate Co‐transporting Polypeptide Inhibitors
Authors: Van Brantegem Pieter , Castillo Yunierkis Perez , Froeyen Mathy , Meuleman Philip , Annaert Pieter
Published: April 2020

Objective

Chronic hepatitis B virus (HBV) infection is responsible for 50% of the cases of hepatocellular carcinoma. As currently available treatment is not able to eradicate the virus, new therapeutic approaches are highly needed. Sodium taurocholate co‐transporting polypeptide (NTCP), a bile acid transporter at the sinusoidal side of hepatocytes, has been identified as receptor for HBV entry into the cell. Since HBV shares a common docking site with NTCP substrates, inhibition of NTCP‐mediated transport is an attractive strategy to inhibit HBV entry and has already been proven successful (e.g. Myrcludex). The aim of this research was to identify novel NTCP inhibitors in vitro and develop a chemometric model that allows virtual screening.

Methods

Wild‐type and NTCP‐transfected Chinese hamster ovary cells were cultured and seeded in black 96‐well plates with transparent bottom. They were preincubated for 15 minutes with a potential inhibitor and the reaction was initiated by adding the fluorescent NTCP substrate tauro‐nor‐THCA‐24‐DBD. The final concentration of compound and substrate was 3 μM. Uptake was determined using fluorescence spectroscopy (excitation 440 nm and emission 580 nm). Potency of the hits was determined by incubating at least seven concentrations of each hit in presence of 3 μM substrate. IC50 values were estimated by fitting the sigmoid Emax model in R version 3.5.1. The inhibitors were subsequently docked into the taurocholate binding site of NTCP and a chemometric model was developed using rapid overlay of chemical structures.

Results

We assessed more than 2500 compounds for their ability to inhibit NTCP. Forty‐one compounds were able to reduce the uptake to less than 50% (of the control, i.e. only substrate present) and were therefore classified as hits. The pIC50 of 30 compounds could be determined and ranged from 3.6 to 6.8. Several rings form the core of the 3D overlay model, surrounded by hydrophobic groups as well as hydrogen bond donor and acceptor groups. While the latter appear to be exposed to the solvent, many of the hydrophobic groups are buried inside the taurocholate binding site.

Conclusions

We identified NTCP inhibitors in vitro and developed a chemometric model that can be used for virtual screening. This model is currently being applied to the ChEMBL database for in silico identification of NTCP inhibitors. Future work also includes in vitro confirmation of the in silico hits.

Support or Funding Information

This research was supported by a grant of the Research Foundation Flanders (FWO grant G089515N).
Stimulation of 5‐HT1F Receptor Increases Vascular Recovery Following Ischemia/Reperfusion‐Induced AKI
Authors: Dupre Tess V. , Janda Jaroslav , Schnellmann Rick G.
Published: April 2020

Acute kidney injury (AKI) is a disease with no treatment. After AKI, there is a marked reduction in renal vasculature. Thus, promoting vascular recovery following AKI could facilitate renal repair as the vasculature is responsible for carrying oxygen and nutrients to extravascular tissues. Our laboratory has shown that stimulating mitochondrial biogenesis (MB) through the 5‐HT1F receptor stimulates recovery from AKI and 5‐HT1F receptor knockout mice have decreased MB and poor renal recovery. Importantly, we have shown that stimulation of the 5‐HT1F receptor with LY344864 or lasmiditan induces MB, branching morphogenesis, and migration in human renal glomerular endothelial cells (EC) in vitro. We hypothesize that the 5‐HT1F receptor plays a role in renal vascular recovery following AKI. 5‐HT1F receptor KO mice have decreased CD31+ and aSMA+ ECs in the kidney, suggesting that the 5‐HT1F receptor plays a role in renal vascular homeostasis. To assess the role of the 5‐HT1F receptor in renal vascular injury, WT and 5‐HT1F receptor KO mice were subjected to IR‐induced AKI. 5‐HT1F receptor KO mice exhibited increased renal Evans blue dye (EBD) extravasation in comparison to I/R injured WT mice 24h following injury, suggesting increased vascular injury. To assess the role of 5‐HT1F receptor in renal vascular recovery following IR‐induced AKI, WT mice were treated with LY344864 (2 mg/kg) beginning 24h after I/R‐induced AKI. LY344864 decreased renal EBD extravasation 144h following injury compared to vehicle treated AKI mice, suggesting that stimulation of 5‐HT1F receptor increases vascular recovery. We also assessed whether or not lasmiditan, a more specific and potent 5‐HT1F receptor agonist which has recently been FDA approved, would induce MB in vivo. Mice were treated with lasmiditan (0.3 mg/kg) for 24h had increased protein levels of PGC1a and TFAM, both of which are regulators of MB and markers of MB. Taken together, these data indicate a role for 5‐HT1F receptor signaling in renal vascular injury and repair following I/R‐induced AKI. We propose that inducing MB in ECs after AKI restores vascular function and stimulates renal repair and recovery, and that repurposing lasmiditan as a treatment option for AKI may prove useful.

Support or Funding Information

This work was supported by
F32 DK120222‐01A1 to TVD (National Institute of Health) and
1BX000851 (Department of Veterans Affairs) to RGS.
Functional in vivo characteristics of antibodies generated by an oxycodone vaccine
Authors: Raleigh Michael D. , King Samantha J. , Saykao Amy T. , Baruffaldi Federico , Gradinati Valeria , Winston Scott , Pentel Paul R. , Pravetoni Marco
Published: April 2020

An oxycodone vaccine (OXY‐sKLH) is being developed to treat opioid use disorder. OXY‐sKLH has been demonstrated to be effective at blocking clinically relevant doses of oxycodone and reducing its reinforcing efficacy. To further assess the potential of OXY‐sKLH for clinical use, critical in vivo properties of the vaccine were studied. Sprague Dawley rats were vaccinated with OXY‐sKLH or control vaccine (sKLH alone) in alum adjuvant. Exposure of vaccinated rats to oxycodone weekly confirmed the expectation that oxycodone alone does not boost oxycodone‐specific antibody titers in vaccinated rats. Re‐vaccination with OXY‐sKLH was necessary to maintain titers. In a separate experiment, antibody titers of vaccinated rats showed a titer of 1.6 × 105 after the final vaccine dose which declined to 0.54 × 105 at 3 months. Vaccine specificity for oxycodone was assessed in vivo. Vaccination protected against oxycodone‐induced, but not methadone‐induced, antinociception. Administration of naloxone to vaccinated rats that had received methadone partially restored nociception and fully reversed respiratory depression. Vaccination of rats with OXY‐sKLH did not alter the pharmacokinetics or antinociception of subsequently administered fentanyl. These in vivo data confirm the previously reported in vitro specificity of this vaccine for oxycodone. Finally, vaccination with OXY‐sKLH extended oxycodone’s half‐life from 0.75 hr in control rats to 24 hr. Taken together, these results indicate that antibody titers generated by OXY‐sKLH are long lasting, and will not interfere with clinically useful non‐targeted opioids, and will decline even in the face of continued oxycodone exposure unless boosted by re‐vaccination. The prolonged oxycodone half‐life in vaccinated rats suggests the potential for antibody saturation if oxycodone exposure continues and is substantial and persistent.

Support or Funding Information

This work was supported by the National Institutes of Health National Institute on Drug Abuse [Grant U01DA038876] (to M.P. and P.R.P.)
Three‐dimensional Visualization of Spatial Relationships Between Coronary Veins and Arteries.
Authors: Tyrak Kamil Wojciech , Bugala Kamil , Bednarz Wojciech , Batko Jakub , Lis Maciej , Holda Jakub Piotr , Witowski Jan , Holda Mateusz Krystian
Published: April 2020

Knowledge of the mutual relationship between the main arteries and veins of the heart is crucial for the safety and effectiveness of interventional and electrophysiological procedures. Thus, the aim of this study was to assess the spatial relationships of main coronary blood vessels using virtual three‐dimensional visualizations. Seventy patients that underwent contrast‐enhanced electrocardiogram‐gated multi‐slice computed tomography of the heart were investigated. Coronary vasculature was segmented and visualized in three dimensions using semi‐automatic algorithms (Mimics Innovation Suite 22.0, Materialise). Right coronary artery dominance was identified in 81.4%, whereas left artery dominance in 11.4% and codominance in 7.1%. The coronary sinus, great cardiac vein and the middle cardiac vein were observed in all patients. Within the inferior interventricular sulcus, the artery was mainly located on the right side of the middle cardiac vein (87.1%), in 58.6% of cases these vessels crossed each other, mostly in their distal part. The coronary sinus was accompanied by the left circumflex artery in 88.6%, but their mutual course was highly variable. In 42.3% the circumflex artery crossed coronary sinus once, in 32.9% twice and in 2.9% three times. The left anterior descending artery was located on the right side of the great cardiac vein in 90.0%, in 51.1% these vessels crossed each other, mainly in middle sector of anterior interventricular sulcus. In conclusion, main coronary arteries and veins cross each other on many levels. Three‐dimensional pre‐procedural coronary vessels visualization is useful tool that may help to plan the procedure and increase its safety and efficiency.
Evaluation of activation of HSF and UPR protein folding pathways by chemical stressors using a yeast reporter system for a hypothesis driven laboratory exercise
Authors: Buckley Martin , Hrizo Stacy , Lama Nawang , Lackner Emily
Published: April 2020

Heat shock factor (HSF) is a transcription factor that is activated in response to an accumulation of misfolded proteins in the cytoplasm. In contrast, the unfolded protein response (UPR) is a transcriptional response pathway that responds to the accumulation of misfolded proteins in the endoplasmic reticulum. Both pathways, when activated by misfolded proteins, result in a transcriptional response that helps the cell to survive by making factors to help the cell sequester and destroy the misfolded proteins. Using aseptic techniques, Saccharomyces cerevisiae with UPR or HSF Beta‐galactosidase reporter plasmids were grown and tested with various doses of 4 different chemical stressors (Dithiothreitol, Diamide, Hydrogen Peroxide, Beta‐Mercaptoethanol) that may induce protein misfolding. Beta‐galactosidase assays were completed to determine the transcriptional response of the HSF and UPR pathways to the chemical stressors. Importantly, this activity emphasizes the mechanism of action of two different cell signaling pathway and demonstrates the use of a marker gene to determine transcriptional activity of a pathway. This laboratory exercises engages the student in critical thinking and data analysis skills as they evaluate data to determine if their chemical stressor activated both pathways or only one pathway.
Oxytocin as a regulatory neuropeptide in the trigeminovascular system: localization, expression and function of oxytocin and oxytocin receptors
Authors: Krause Diana N. , Warfvinge Karin , Grell Anne-Sofie , Edvinsson Jacob , Haanes Kristian A. , Edvinsson Lars
Published: April 2020

Recent clinical findings show oxytocin (OT) inhibits migraine headache, suggesting an unexpected role of this neuropeptide/hormone in trigeminal pain signaling. Here we show OT receptors (OTR) are extensively expressed throughout the rat trigeminovascular system. Using immunohistochemistry and qPCR, we found OTR expression in trigeminal ganglia (TG), in particular, in a large portion of the A‐delta sensory neurons and fibers. OTR also was expressed in the central target of TG afferents, the trigeminal nucleus caudalis. A small number of C‐fiber sensory neurons in the TG expressed OTR; which was colocalized with the neuropeptide CGRP (calcitonin gene related peptide). However, OT had no effect on K+‐evoked CGRP release from either isolated TG or TG afferents in the dura mater. Another peripheral TG target, the cranial arteries, were found to constrict in response to OT in vitro; an effect blocked by the OTR antagonist L368899. OT immunoreactivity was found in TG satellite glial cells but OT mRNA was undetectable in TG. Thus OT from the circulation most likely acts on OTR in the TG to affect pain transmission. These effects may help explain hormonal influences in migraine and offer a novel target for treatment.

Support or Funding Information

Lundbeck Foundation and Swedish Medical Research Council
Role of AVP in the Pathology of Liddle’s Syndrome
Authors: Mironova Elena , Soares Antonio Garcia , Xiang Hong , Gurley Susan , Beermann Edith , Stockand James David , Pao Alan Chun-Yao
Published: April 2020

The epithelial Na+ channel, ENaC, is expressed in the apical plasma membrane of renal principal cells. ENaC activity is rate limiting for sodium reabsorption across the distal nephron and consequently, is the final arbiter of renal sodium excretion. Gain of function mutations in ENaC cause Liddle’s syndrome: an autosomal dominant form of inheritable hypertension. The hypertension of Liddle’s syndrome results from uncontrolled sodium reabsorption via hyperactive ENaC. We hypothesized that improper sodium reabsorption during Liddle’s syndrome may elevate plasma tonicity enough to evoke release of vasopressin (AVP). Indeed, AVP levels are elevated in Liddle’s mice, which have increased ENaC activity as assessed by patch clamp electrophysiology of isolated CNT/CCD. Tolvaptan, an AVP V2 receptor blocker, decreased ENaC activity in Liddle’s mice. Exogenous desmopressin (DDAVP), a V2 receptor specific agonist, had no further effect on ENaC activity in Liddle’s mice. However, DDAVP further increases blood pressure in Liddle’s mice, and this high blood pressure decreases with administration of benzamil, an ENaC blocker. We conclude from these results that ENaC activity is saturated in Liddle’s mice, in part due to elevated AVP levels, but that saturated ENaC activity is still necessary for additional effects of DDAVP on blood pressure in the Liddle’s model. Because DDAVP increases blood pressure but not ENaC activity in Liddle’s mice, we speculate that additional DDAVP‐stimulated water reabsorption further increases blood pressure in this model. Thus, in the Liddle’s mouse, while ENaC activity is saturated, water reabsorption can still respond to additional AVP signaling. Experiments investigating DDAVP effects on AQP2 and AQP3 expression are ongoing and will test whether the hyperosmotic draw of solute from tubular fluid across the collecting duct is saturated in the Liddle’s mouse.

Support or Funding Information

This research was supported by NIH/NIDDK grants R01DK113816 to JDS and R01DK103758 to AP and JDS.
Calcium‐Sensing Receptor function regulation by hyaluronan
Authors: Lazrak Ahmed , Yu Zhihong , Matalon Sadis
Published: April 2020

Mice exposure to halogens, chlorine (Cl2) or bromine (Br2), develop increased airway resistance and airway hyper‐responsiveness (AHR) to methacholine, which lasted for at least one‐week post exposure. Herein, we investigated the mechanisms involved. Mice exposed to halogens (600 ppm Br2 or 400 ppm Cl2) for 30 min developed AHR 24 hours post exposure. Nifedipine, an L‐type calcium channel blocker, administered subcutaneously post halogens exposure, resulted in higher AHR compared to halogens alone. In contrast, diltiazem (a non‐dihydropyridine L‐type calcium channel blocker) reversed halogens induced AHR. Exposure of human airway smooth muscle Br2 100 ppm for 10 min) resulted in significant membrane depolarization (Vm Air: 62±3 mV; 3 h post halogens −45±5 mV; mean±1 SEM; p<0.001), increased intracellular calcium [Ca2+]i ( < 0.5 μM) and the expression of the Calcium‐Sensing Receptor (Ca‐SR). All of these effects were exacerbated with treatment with nifedipine but not diltiazem. Treatment of hASMCs with an siRNA against Ca‐SR significantly reduced nifedipine induced Vm depolarization and [Ca2+]i increase. Intranasal administration of an antagonist to Ca‐SR in vivo (NPS2143, 1 μM) reversed the effects of halogens and nifedipine on AHR. Incubation of hASMCs with Low Molecular Weight Hyaluronan (LMWHA), generated by exposing High Molecular Weight Hyaluronan (HMWHA) to halogens, induced Vm depolarization, [Ca2+]i increase, and Ca‐SR expression analogously to halogens. Addition of HMWHA to cells exposed to halogens, or incubated with LMWHA, reversed these effects in vitro, and improved AHR in vivo. We conclude that injurious effects of halogens on AHR are mediated by the induction of Ca‐SR over‐expression in the peripheral airway smooth muscle cells.

Support or Funding Information

5UO1 ES026458 03; 3UO1 ES026458 03S1; 5UO1 ES027697 02
Human Keratinocytes Synthesize Ultra Long‐Chain Sphingoid Bases (Up To C28) That Are Abundant in Skin Stratum Corneum
Authors: Berdyshev Evgeny , Bronova Irina , Bronoff Anna S. , Goleva Elena , Leung Donald Y.M.
Published: April 2020

Skin sphingolipids are unique and fulfill the important task of forming a hydrophobic barrier to limit water loss from the body but also to protect from allergen, bacterial and viral penetration through the skin. Skin sphingolipids are highly diverse in their combination of fatty acids and sphingoid bases, the presence of highly hydrophobic ω‐esterified ceramides (EOS‐CER), and the abundance of ultra long‐chain fatty acids (ULCFA, up to C36) within ceramide molecules. The chain length of sphingoid bases in ceramides is commonly attributed to C18 molecules but there are only a few reports demonstrating the presence of C20‐ and C22‐sphingoid bases in skin ceramides. As free sphingoid bases and ceramides are metabolically connected, it is assumed that similar to other tissues, C18‐sphingoid bases represent the majority of sphingoid bases within the total pool of free and ceramide‐linked molecules in the skin.

Our sphingolipidomic work during the study of the mechanisms leading to the impairment of skin barrier function has revealed that ceramides with C18‐, C20‐, and C22‐ sphingoid bases are almost equally represented in human skin stratum corneum. Surprisingly, we found that free sphingoid bases in stratum corneum are highly abundant and composed primarily of ultra long‐chain (C24‐ and C26‐) dihydrosphingosines. This finding prompted us to investigate in details the ability of skin keratinocytes to synthesize ultra long‐chain length sphingoid bases. Using undifferentiated and Ca2+‐differentiated primary human keratinocytes, [U‐13C,15N]‐serine, and stable isotope pulse‐labeling approach in vitro, we have confirmed that all ceramides and free sphingoid base molecules do incorporate the labeled serine into their structure. Next, we explored the ability of serine palmitoyltransferase (SPT), the enzyme that initiates sphingolipid biosynthesis, to synthesize ultra long‐chain sphingoid bases in vitro. By comparing the activity of SPT in total cell lysates prepared from undifferentiated and Ca2+‐differentiated human keratinocytes, 16:0–24:0‐Coenzyme A molecules and [U‐13C,15N]‐serine, we confirmed the ability of SPT expressed in human keratinocytes to produce, albeit at low capacity, C24‐ and C26‐3‐keto‐dihydrosphingosines. As keratinocytes and skin stratum corneum do not have measurable amounts of ceramides with C24‐C26‐dihydrosphingosines or C24‐C26‐sphingosines, we conclude that keratinocyte ceramide synthases are unable to utilize dihydrosphingosines with carbon chain length more than C22, thus providing a foundation for the accumulation of C24‐C26‐dihydrosphingosine in human stratum corneum. The question of the possible special biological function of long‐chain dihydrosphingosines in human skin is currently under investigation.
Role of the Innate Immune System in the Development of Hypersensitivity Pneumonitis
Authors: Ocasio-Rivera Marinel , Elmore Jessica , August Avery
Published: April 2020

Hypersensitivity pneumonitis (HP) is an extrinsic allergic alveolitis disease that is constantly affecting the farmers. It is triggered by the continuous exposure of environmental antigens, specifically antigens that grow in hot humid conditions in hay like Saccharopolyspora rectivirgula (SR). While a role of T cells has been established in the development of this diseases, the role of the innate immune system is still largely unknown in the development of lung inflammation leading to HP. To study this disease, we used a mouse model of HP, induced by SR, along with Rag−/− mice which only have an innate immune system and lack an adaptive immune system (and thus have no T cells). We hypothesized that the development of Hypersensitivity pneumonitis requires the presence of T (and B) cells, at the same time, there will be a lower percentage and number of inflammatory cells in the lungs of Rag−/− mice upon exposure to SR. WT and Rag−/− mice were exposed to SR. Lungs, spleen and lymph nodes where collected to analyze their response. We examined the percentage and number of myeloid and Innate Lymphoid Cells (ILC), as well as their ability to produce cytokines. We found that there was no significant difference in the number of Innate Lymphoid cells and myeloid cells between SR‐exposed WT and Rag−/− mice. These results may be due to the low concentration of bacteria used for these experiments. In future experiments, we propose to use a higher concentration of SR to induce inflammation.

Support or Funding Information

This work was supported in part by grants from the NIH‐AI38570, NIH‐AI20701, and NIH‐AI129422. NIH‐NIGMS #2R25GM096955 (RISE). VIP‐Veterinary Investigation Program.
Canagliflozin provides cardioprotection and renoprotection against isoprenaline‐induced oxidative stress by stimulating AMPK‐Akt‐eNOS‐NO signaling axis
Authors: Hasan Raquibul , Lasker Shoumen , Hasan Ahasanul , Zamila Mushfera , Zerin Farzana , Chowdhury Faizul Islam , Rahman Md. Mizanur , Khan Ferdous , Subhan Nusrat , Alam Md. Ashraful
Published: April 2020

The antidiabetic drug canagliflozin, a SGLT2 inhibitor, has been reported to possess protective effects against ischemia‐reperfusion and chemically‐induced cardiac and renal injuries. However, to our knowledge, no studies have investigated the protective effects of canagliflozin in isoprenaline (ISO)‐induced cardiac and renal oxidative damage – a model mimicking sympathetic nervous system (SNS) overstimulation‐evoked organ injuries in humans. Therefore, we investigated effects of canagliflozin in ISO‐induced cardiac and renal oxidative stress, and their underlying molecular mechanisms. Our tissue biochemistry, histology, and Western blotting data showed that ISO administration inflicted pro‐oxidative, pro‐inflammatory and pro‐apoptotic changes in the heart and kidney tissues. In contrast, treatment with canagliflozin at 5 mg/kg body weight attenuated levels of cardiac and renal oxidative stress markers including malondialdehyde (MDA), advanced protein oxidation product (APOP), myeloperoxidase (MPO) and nitric oxide (NO), and restored levels of endogenous antioxidant molecules such as catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH) that were depleted in ISO‐treated rats. Western blot analysis revealed that canagliflozin increased activity of anti‐oxidant/anti‐inflammatory proteins such as AMP‐activated protein kinase (AMPK), Akt and eNOS, and decreased pro‐oxidative/pro‐inflammatory proteins including iNOS and NADPH oxidase isoform 4 (NOX4). Protein expression analysis further revealed that ISO increased the ratio of pro‐apoptotic/anti‐apoptotic protein (Bax:Bcl‐2) expression, which was significantly reduced by canagliflozin treatment. Our histological examination demonstrated that in the heart and kidney tissue sections, canagliflozin attenuated ISO‐induced histopathological changes, including inflammatory cell infiltration, collagen deposition, and fibrosis. Consistent with our biochemical data, canagliflozin augmented heart and kidney functions in ISO rats, further supporting a protective role of this drug in ISO‐induced oxidative stress. In summary, we show that canagliflozin produces cardioprotective and renoprotective actions via suppression of iNOS, NOX4 and Bax, and activation of AMPK‐Akt‐eNOS‐NO signaling axis.

Support or Funding Information

This work was supported by a start‐up grant from the Mercer University College of Pharmacy to Dr. Raquibul Hasan and a research grant from North South University to Dr. Md Ashraful Alam