bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationThis 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 InformationPT‐A has been funded by Foundation Ramon Areces. This work has been supported by grant ZIABC011691 to J.B.
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 InformationZoe 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

ObjectiveChronic 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.MethodsWild‐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.ResultsWe 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.ConclusionsWe 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 InformationThis 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 InformationThis 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 InformationThis work was supported by the National Institutes of Health National Institute on Drug Abuse [Grant U01DA038876] (to M.P. and P.R.P.)
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 InformationLundbeck 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 InformationThis 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 Information5UO1 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.