bluuebunny/crossref_metadata_2025 huggingface dataset:
Human calcaneal external shape relative to activity and foraging levels
Authors: Harper Christine M. , Ruff Christopher B. , Sylvester Adam D.
Published: April 2020

The role of the calcaneus in weight‐bearing suggests that it may reflect differences in activity and foraging levels among different modern human populations. While lower limb cortical and trabecular bone have been demonstrated to vary with regard to human behavior, human calcaneal variation has not been studied in this context. Here we analyze the entire calcaneal external shape of recent industrialized modern humans (n=60), Native American agriculturalists (n=47), a transitional Native American population that had not fully adopted agriculture (n=10), and Late Stone Age Khoesan hunter‐gatherers (n=13).

Calcanei were either surface or micro‐CT scanned and their external shape analyzed using a three‐dimensional geometric morphometric sliding semilandmark analysis. Semilandmarks were allowed to slide relative to an updated Procrustes average to minimize the bending energy of the thin plate spline interpolation function. Final landmark configurations were aligned using a Generalized Procrustes Analysis. Shape variation was summarized using a principal components analysis. Procrustes distances between different human populations were calculated, and resampling statistics run to test for significant differences.

All human populations demonstrate statistically different calcaneal morphologies (p<0.005 for all comparisons). Industrialized and non‐industrialized populations separate from each other across the first six principal components (52.34% of variance). Non‐industrialized populations demonstrate a mediolaterally wider and superoinferiorly taller posterior calcaneus and tuber, as well as a larger peroneal trochlea. This greater robusticity is likely adapted for the increased load through the calcaneus associated with a non‐sedentary lifestyle. The industrialized sedentary modern humans, however, possess a relatively larger lateral plantar process, potentially as a compensatory mechanism for bipedal behaviors in a generally less robust and more antero‐posteriorly elongate calcaneus.

Support or Funding Information

This project was supported by NSF grant # BCS ‐ 1824630.
Flux Of Glucose Through Pentose Phosphate Pathway And Glycolysis In Mouse Hearts Subjected To Low‐Flow Ischemia
Authors: Fan Wei , Lu Daning , Shi Xinwen , He Huamei
Published: April 2020

Introduction

The molecular events that lead to irreversible myocardial damage during severe ischemia remain poorly understood. Changes in cytosolic redox status and ability of mitochondria to oxidatively metabolize fatty acids and carbohydrates have been implicated in ischemic Injury. The pentose phosphate pathway (PPP) produces NADPH, which can be used to maintain glutathione in its reduced state, providing protection against oxidative damage. The rate of glycolysis during ischemia determines extent of ischemic injury. We investigated flux of glucose through PPP and glycolysis in mouse hearts subjected to low‐flow ischemia in this study.

Methods

Isolated mouse hearts were perfused in Langendorff mode with 10mM [1,2‐13C2]‐glucose for 60min, followed by 90min low‐flow ischemia (perfused at 10% of baseline flow rate). The cardiac function was recorded with PowerLab Data Acquisition Systems. Lipid peroxidation was determined with Colorimetric/Fluorometric Assay Kit. The flux of glucose through the PPP and glycolysis was assessed by the contribution of the doublet (D23, [2,3‐13C2]‐lactate) and singlet (S3, [3‐13C]‐lactate) to the total 13C‐NMR signal of lactate C3 in effluent, respectively.

Results

90min low‐flow ischemia resulted in both systolic dysfunction (as indicated by decreased left ventricular systolic pressure, rate of tension development and developed pressure) and diastolic dysfunction (as indicated by increased end diastolic pressure and decreased rate of relaxation) accompanied with increased lipid peroxidation. Furthermore, the flux of glucose through PPP increased substantially and correlatively with that through glycolysis.

Conclusion

[1,2‐13C2]‐glucose can be used to simultaneously assess flux of glucose through PPP and glycolysis. The flux of glucose through both PPP and glycolysis increased during myocardial low‐flow ischemia.

Support or Funding Information

This study was supported by Department Fund.
The Histone Methyltransferase G9a Promotes Cholangiocarcinogenesis through Regulation of the Hippo Pathway Kinase LATS2 and YAP Signaling Pathway
Authors: Ma Wenbo , Han Chang , Zhang Jinqiang , Song Kyoungsub , Chen Weina , Kwon Hyunjoo , Wu Tong
Published: April 2020

Background

Cholangiocarcinoma (CCA) is a highly malignant epithelial tumor of the biliary tree with poor prognosis. A better understanding of the molecular mechanisms of cholangiocarcinogenesis is of paramount importance to develop more effective target therapy.

Methods

qRT‐PCR and Western blotting analyses were used to determine the expression of specific mRNAs and proteins. ChIP‐PCR was performed to analyze the enrichment of G9a and H3K9me2 in the promoter region of LATS2 gene. Dual‐Luciferase reporter assay system was used to evaluate YAP luciferase activity. CCA xenograft was developed by inoculation of human CCA cells with or without G9a depletion into SCID mice. CCA tumor induction was developed by SB transposase‐mediated integration of oncogenes (NICD/Akt) to mice via hydrodynamic tail vein (HDTV) injection.

Results

We present novel evidence that the histone‐lysine methyltransferase G9a is upregulated in human CCA and that G9a enhances CCA cell growth and invasiveness through regulation of the Hippo pathway kinase LATS2 and YAP signaling pathway. Kaplan–Meier survival analysis revealed that high G9a expression is associated with poor prognosis of CCA patients. In experimental systems, depletion of G9a by siRNA/shRNA or inhibition of G9a by specific pharmacological inhibitors (UNC0642 and UNC0638) significantly inhibited human CCA cell growth in vitro and in SCID mice. Increased G9a expression was also observed in mouse CCA induced by hydrodynamic tail vein injection of NICD and myr‐Akt. Administration of the G9a inhibitor UNC0642 to NICD/Akt‐injected mice reduced the growth of CCA, in vivo. These findings suggest that G9a inhibition may represent an effective therapeutic strategy for the treatment of CCA. Mechanistically, our data show that G9a‐derived dimethylated H3K9 (H3K9me2) silenced the expression of the Hippo pathway kinase LATS2 and this effect led to subsequent activation of oncogenic YAP. Accordingly, out data showed that G9a depletion or inhibition reduced the level of H3K9me2 and restored the expression of LATS2 leading to YAP inhibition.

Conclusion

Our findings provide novel evidence for an important role of G9a in cholangiocarcinogenesis through regulation of LATS2‐YAP signaling and suggest that this pathway may represent a potential therapeutic target for CCA treatment.
Functional analysis of mosquito and Drosophila Na+‐dependent cation‐chloride cotransporters
Authors: Gillen Christopher M. , Holmes Heather L. , Piermarini Peter M. , Romero Michael F.
Published: April 2020

Dipteran Na+‐dependent cation‐chloride cotransporters (CCCs) group into two clades. Drosophila melanogaster Ncc69 is a bona fide Na+‐K+‐Cl− cotransporter and has a single ortholog in Aedes aegypti (aeNKCC1). Drosophila Ncc83 has not been physiologically characterized and has two orthologs in Ae. aegypti (aeCCC2 and aeCCC3). Previous work showed that oocytes expressing aeCCC2 have higher Na+ conductances than controls. Moreover, in flux assays using Li+ as a Na+ tracer, Li+ transport by aeCCC2 was not dependent upon extracellular Cl− and not inhibited by the loop diuretics furosemide or bumetanide. These are surprising results, since previously characterized CCCs are electroneutral, Cl−‐dependent, and sensitive to the loop diuretics. We have further characterized aeCCC2 and performed an initial analysis of Ncc83, using two‐electrode voltage clamp. Following a hypotonic pre‐incubation, changes in membrane potential upon Na+ replacement were 20 ± 3 mV in Ncc83 and 26 ± 4 mV in aeCCC2, approximately two‐fold greater than before swelling. Na+‐dependent currents in oocytes expressing aeCCC2 depended on extracellular Na+ with saturable Michaelis‐Menton kinetics. The apparent affinity for Na+ was 4.6 ± 0.5 mM (mean, SEM, n=5). To characterize the function of Ncc83 in Drosophila Malpighian tubules (MT), we developed procedures to assess the cation concentrations of secreted fluid in Ramsay secretion assays using cation‐exchange chromatography. The physiological cations were readily detectable in 10,000–50,000 fold dilutions of secreted fluid, and Li+ and Rb+ were also detectable when added to the bathing saline as tracers for Na+ and K+. In w118‐control flies, fluid secreted by MT measured using these procedures had a [K+] of 104.1 ± 8.8 mM (mean, SEM, n=7) and [Na+] of 61.2 ± 7.8 mM. In preliminary experiments, [K+] was 112.7 ± 1.3 mM (n=4) and [Na+] was 48.2 ± 4.1 mM in fluid secreted by MTs of flies with MT‐specific knockdown of Ncc83 using the GAL4‐UAS RNAi system. Further work is needed to determine the physiological roles of Ncc83 and aeCCC2. Funding: American Physiological Society Research Career Enhancement Award, NIH F33 GM131599, and Kenyon College.

Support or Funding Information

Funding: American Physiological Society Research Career Enhancement Award, NIH F33 GM131599, and Kenyon College.
Expression of genes related to autophagy and protein breakdown are positively correlated with protein synthesis and protein breakdown in skeletal muscle of healthy adults after a bout of resistance exercise
Authors: Wu Hexirui , Kim Il-Young , Jang Ji-Woong , Ferrando Arny , Wolfe Robert , Baum Jamie
Published: April 2020

While it is well‐established that exercise increases muscle protein synthesis rate, it is still unclear that what role of protein breakdown plays in muscle after exercise. Therefore, the objective of this study was to determine if rates of protein synthesis and breakdown correlate with expression of genes related autophagy and protein degradation in skeletal muscle of healthy adults after a bout of resistance exercise. This is a secondary analysis of a previous published study (
Kim et al., 2016). In total, twenty‐three subjects were divided into two groups, rest (n = 12) and exercised (n = 11). Real‐time PCR was used for analyzing gene expression of baseline muscle samples from both groups, and student t test was used to compare significance (p < 0.05) between two groups. Relationships between variables were assessed using Pearson’s Product‐Moment Correlation, and p value lower than 0.05 was considered as significant. Autophagy‐related genes p62, p53, ATG3, ATG5, LC3B, Beclin1, ATF4, and GADD54A, were significantly upregulated (p < 0.05) in skeletal muscle from exercised subjects versus subjects at rest. Two genes involved in ubiquitin‐proteasome pathway, MuRF1 and Atrogin1, were also upregulated (p < 0.05) after exercise. Neither protein synthesis nor protein breakdown rate was correlated with gene expression in skeletal muscle from subjects at rest. However, protein synthesis rate was positively correlated (p < 0.05) with increased expression of p62, p53, ATG3, ATG5, ATG7, LC3B, Beclin1, ATF4, GADD54A, Atrogin‐1 and MuRF1 in exercised muscle. Protein breakdown rates were positively associated (p < 0.05) with increased expression of ATF4, ATG5, and p62 in exercised skeletal muscle. In conclusion, the results provide preliminary evidence that genes related to autophagy and protein breakdown are upregulated after exercise in skeletal muscle from healthy adults and are positively correlated with muscle protein synthesis rate.

Support or Funding Information

This study is funded by an Arkansas Biosciences Institute grant, we would like to thank thegenerous contribution of muscle samples by Dr. Wolfe.
Leptin Receptor Blockade Abolished Hypertension and Decreased Hypoxic Ventilatory Response in a Model of Obesity‐Related Hypertension
Authors: Kim Lenise Jihe , Shin Mi-Kyung , Pho Huy , Otvos Laszlo , Tufik Sergio , Andersen Monica Levy , Polotsky Vsevolod Y.
Published: April 2020

INTRODUCTION

Obesity causes hypertension and exacerbates sleep disordered breathing. Leptin is an adipocyte produced hormone which is implicated in regulation of metabolic rate, food intake, control of breathing and blood pressure. Obese individuals are resistant to metabolic and respiratory effects of leptin, but appeared to be sensitive to hypertensive effects of the hormone. We hypothesized that New Zealand obese mice (NZO), which are resistant to metabolic effects of leptin, remain susceptible to cardiovascular, but not respiratory, effects of this hormone.

METHODS

Male NZO mice of 12–13 weeks‐old were treated with leptin receptor blockers Allo‐aca (n=5) or control peptide Gly11 (n=3) for 9 consecutive days. Doses of 0.2mg/kg were administered 2x/day, at 10AM and 6PM subcutaneously. Blood pressure was measured by telemetry. Ventilation was assessed by whole‐body barometric plethysmography and control of breathing was examined by assessing the hypoxic ventilatory response (HVR). Sleep studies were conducted by full‐polysomnography.

RESULTS

Systemic leptin receptor blocker Allo‐aca did not affect body weight and food intake in NZO mice. In contrast, it induced a decrease in the mean arterial blood pressure from 132.9±5.9 mmHg to 121.2±11.3 mmHg during the light phase (P<0.001). Allo‐aca did not affect baseline minute ventilation in awake mice, but decreased HVR (P<0.05). There was a trend for systemic blockade of leptin receptors with Allo‐aca to suppress minute ventilation during NREM and REM sleep, whereas the control peptide Gly11 had no effect.

CONCLUSIONS

NZO mice were resistant to metabolic effects of leptin, but they were sensitive to the hypertensive and respiratory effects of the hormone. Thus, leptin receptor blockers can be used for pharmacotherapy for obesity‐associated hypertension, but sleep‐related hypoventilation should be considered as a potential side effect.

Support or Funding Information

NIH R01s HL128970 and HL133100 to Dr. Vsevolod Y. Polotsky. Dr. Tufik and Dr. Andersen are CNPq fellowship (Brazil) recipients. Dr. Lenise Jihe Kim was supported by Associação Fundo de Incentivo à Pesquisa (AFIP, Brazil).
Regulation of the Mannitol Locus by MtlR in Vibrio cholerae
Authors: George Micayla , Liu Jane
Published: April 2020

The intake of mannitol, a sugar produced by plants and algae, by Vibrio cholerae increases biofilm formation and thereby persistence of the pathogenic bacterium. The process of mannitol uptake is mediated by the mannitol phosphotransferase system (PTS). Within this system, the protein MtlA is directly responsible for the intracellular transport of mannitol, and is encoded by the mtlA gene located within a group of genes (mtlA, D, and R) on the mannitol (mtl) locus. While it is known that protein MtlR represses the expression of mtlA at the transcriptional level, the mechanism of this repression is unknown. The aim of this project is to better understand the dynamics between MtlR and other protein regulators of the mtl locus, such as the CRP activator protein, to gain insight into the mechanism of MtlR regulation of mtlA expression. This was accomplished through assessment of MtlR and CRP levels across V. cholerae via Western Blots, as well as through measuring mtlA expression in the absence of CRP and/or MtlR via LacZ assays. Previous work has shown that MtlR levels fluctuate depending on available carbon source, but we found CRP levels to be relatively unaffected. We also found that while deletion of the mtlR gene increases mtlA expression, this is not observed when the crp gene is also deleted, suggesting that MtlR repression is secondary to CRP activation of mtlA expression. This work helps shed light on possible mechanisms by which MtlR might be negatively regulating the expression of mtlA and overall intracellular mannitol intake by V. cholerae.

Support or Funding Information

National Institutes of Health; The Camille and Henry Dreyfus Foundation
‘Must See’ Videos? Why Educators Need Better Video Analytics to Measure Learning
Authors: Van Nuland Sonya E. , Langley Natalie R.
Published: April 2020

The pervasiveness of flipped classrooms and blended learning has tasked educators with creating content targeting fundamental concepts for asynchronous learning. The visual nature of anatomy subject matter makes video technology particularly well‐suited to convey important relationships. Education technology companies purport that video analytics may provide educators insights into their learners and the effectiveness of their videos. This study examines this claim using analytics recorded by a learning video platform during a 7‐week human anatomy course at the Mayo Clinic Alix School of Medicine (Arizona campus). First‐year medical students (n=48) were provided with daily faculty‐guided videos (n=21) of prosected cadavers [mean length ± SD (mm:ss) = 07:32 ± 3:28]. The videos highlighted structures covered during laboratory sessions. Each video was released less than 36 hours after the associated laboratory session. Video analytic data was recorded, including the number of times a video was viewed and played, average view time and the length of time a student watched a video before closing the page or pausing the video and never restarting it (drop‐off rate). Multiple linear regression analysis was conducted using JMP software to examine the relationship between these video analytic variables and laboratory practical examination performance. None of the video analytic variables, when calculated by individual video or individual student, were significant predictors of practical exam performance [mean exam performance = 88.83%; individual videos, R2 = 0.024, p = 0.78; individual student, R2 = 0.113, p = 0.63]. The results of this study are consistent with previous analyses of the educational effectiveness of videos in anatomy courses, in spite of the company’s claim that “videos can improve learning results”. While videos may make education more accessible and engaging, the impact on student learning is difficult to determine using the basic metrics supplied by the video learning platform. Until more sophisticated video analytics become available, such data will remain of limited use to educational researchers.
After salt: ENaC in self‐sustaining hypertension
Authors: Arkhipov Sergey N. , Potter D’Anna L. , Pavlov Tengis S.
Published: April 2020

Background

Every year thousands of hypertensive patients reduce salt consumption in the efforts to control blood pressure. However, most of the studies report a cohort of patients who does not significantly respond to sodium restriction. Generally, most of the studies agreed that about one‐third of the patients had an excellent response, one‐third had only a modest response, and one‐third had little or no response. Irreversibility of high blood pressure is associated with various mechanisms underlying self‐sustaining character of hypertension. Unfortunately, hypertension causes multiple abnormalities in the body which stays even when the trigger, caused the initial blood pressure raise, is gone. We hypothesize that chronic hypertension leads to a significant renal damage and abnormally high sodium reabsorption.

Methods

We used Dahl salt‐sensitive rats for chronic continuous observation of blood pressure with radiotelemetry in conscious free moving animals. Rats were fed a 4% NaCl diet for 3 weeks to induce hypertension and then diet was switched back to normal (0.4% NaCl). Patch‐clamp analysis was performed on freshly isolated split‐open cortical collecting ducts and CHO cells to characterize activity of the epithelial sodium channel (ENaC), responsible for the sodium reabsorption in the aldosterone‐sensitive distal nephron.

Results

We found that 4% sodium diet significantly increases blood pressure within 3 weeks (from 111±0.9 to 138±3.5 mmHg) and switching back to 0.4% NaCl diet slightly reduces blood pressure (to 133.6±7.6). Patch clamp analysis reveals that development of hypertension was accompanied with elevated ENaC activity which also stayed high (NPo=1.22±0.23, n=8) after the salt challenge. Subsequent blockage of ENaC with a selective inhibitor, benzamil, caused a dramatic drop in blood pressure indicating that ENaC activity contributes to maintain sustaining hypertension in absence of the high salt consumption. We also found significant renal damage caused by hypertension. As earlier studies report that inflammation and reactive oxygen species production are involved in improper renal function and setting high blood pressure, we performed a subset of whole‐cell patch clamp experiments on CHO cells overexpressing mENaC to study effect of hydrogen peroxide on ENaC activity. We found that treatment with 100μM H2O2 within a few seconds to 2 minutes stimulated the ENaC current of the cells (−144±12 pA/pF vs −190±22 pA/pF before and after treatment respectively, n=8, p=0.0078).

Based on our earlier publications and the current data we conclude that ENaC activity contributes to both development of salt‐sensitive hypertension and its continuation in the absence of high salt challenge and reactive oxygen species production can keep ENaC active.

Support or Funding Information

Acknowledgement

ASN Carl W. Gottschalk award; R00 HL116603
A Mathematical Model of the Rat Kidney. II. Antidiuresis
Authors: Weinstein Alan M.
Published: April 2020

Water conservation by the kidney requires a hypertonic medullary interstitium, NaCl in outer medulla (OM) and both NaCl and urea in inner medulla (IM), along with a vascular configuration that protects against washout. A multi‐solute mathematical model of the rat kidney, which includes the important acid‐base species, was developed and used first to examine K+‐induced natriuresis (Am. J. Physiol. 312:F925, 2017). In the present work, model computation has been streamlined by parallelizing the calculation of the model Jacobian, and this has allowed finer medullary spatial resolution, along with more extensive examination of model parameters. Tubule compliance is now represented along the whole nephron, and this attenuates pressure swings due to variations in urine flow. OM NaCl is modestly increased when transporter density in ascending Henle limbs (AHL) from juxtamedullary nephrons (JM), is scaled to match the greater JM nephron solute flow. However, higher NaCl transport produces greater CO2 generation, and by virtue of countercurrent vascular flows, establishment of high medullary pCO2. This CO2 gradient can be mitigated, by assuming that a fraction of medullary AHL and collecting duct transport is powered anaerobically. Reduction of vascular flows or increasing vessel permeabilities does little to further increase OM solute gradients. In contrast to the medullary models of others, vessels in this model all have solute reflection coefficients close to zero; increasing these coefficients provides no substantive enhancement of solute profiles, but does generate high interstitial hydrostatic pressures, which distort tubule architecture. As others have noted, increasing medullary urea delivery via entering vasa recta, enhances IM urea, although not nearly to levels found in rats. In sum, (1) the magnitudes of medullary gradients of Na+, urea, and ammonia are still not captured by the model; and (2) the countercurrent architecture that provides antidiuresis also produces exaggerated pCO2 profiles and appears to be an unappreciated constraint on models of medullary function.

Support or Funding Information

NIH RO1‐DK‐29857
Therapeutic Role of Dietary Nitrates on Cardiorespiratory Function in Cancer Survivors
Authors: Turpin Vanessa-Rose Garcia , Parr Shannon K. , Hammond Stephen T. , Colburn Trenton D. , Ade Carl J.
Published: April 2020

Introduction

The adverse physiological consequences of anticancer therapy include direct injury to the cardiovascular‐skeletal muscle axis. As such, cancer patients are at an increased risk of both cancer therapy‐related and age‐related pathological outcomes, including primary cardiovascular disease, exercise intolerance, and cancer‐related fatigue. To date, however, therapeutic strategies that mitigate these negative effects within the human body have yet to be established. Previous work has demonstrated that dietary nitrate (NO3−) supplementation can improve cardiac, vascular, and cardiorespiratory exercise parameters, highlighting its potential therapeutic use in clinical populations. Therefore, we hypothesized that NO3− supplementation improves both cardiac performance and exercise capacity. We also investigated the effects of NO3− supplementation on estimates of components of the convective oxygen transport pathway to obtain insight into potential mechanisms of action.

Methods

To date, 5 cancer survivors (58.6 ± 11.3 years) with a history of anticancer therapy completed a randomized, double‐blind, crossover study with an acute, single‐dose administration of NO3− or placebo (PL) [140 mL]. Transthoracic echocardiographic measures at rest were made to obtain left ventricular stroke volume. Patients performed a supine‐cycling steady‐state exercise test (30W) and a maximal‐effort cardiopulmonary exercise test, with measurements of arterial blood pressure, stroke volume, and cardiac output.

Results

There was a statistically significant increase in plasma nitrite during the NO3− condition compared to PL (NO3− 1300 ± 963 μM vs. PL 111 ± 49 μM, respectively; P = 0.02). Additionally, we observed a decrease in oxygen uptake (VO2) during steady‐state exercise with NO3− compared to PL (NO3− 8.2 ± 2.4 vs. PL 8.6 ± 2.5 ml/kg/min; P = 0.03), indicating an improved exercise efficiency. Resting and steady‐state arterial blood pressure, stroke volume, and cardiac output were not different between conditions. Furthermore, we did not observe any differences between conditions for VO2 peak (NO3− 22.87 ± 4.14 vs. PL 23.45 ± 4.4 ml/kg/min; P = 0.31) or total work done (NO3− 75.73 ± 29.9 vs. PL 75.82 ± 31.3 kJ; P = 0.48) during the maximal‐effort cardiopulmonary exercise test. Similarly, there was no difference is peak cardiac output, convective O2 transport, or the arterial‐venous O2 difference.

Conclusions

A single‐dose of inorganic nitrate supplementation in cancer survivors with a history of anticancer therapy enhanced steady‐state exercise efficacy, but had no effect on exercise cardiac performance or peak exercise capacity.
Effects of Prior Anatomy Coursework on Assessment Performance and Study Habits
Authors: Wehrman Gregory , Ryan Katharine , Auerbach Benjamin
Published: April 2020

Students entering health science fields often take multiple human anatomy courses throughout their education, including settings such as high school, community colleges/technical schools, and 4‐year undergraduate universities. Additionally, it is not uncommon for students to retake a human anatomy course during their undergraduate careers if they are not satisfied with their initial performance. While research has shown that final grade performance improves for those retaking the same undergraduate course, it is unclear whether previous anatomy coursework in high school, community college, or other undergraduate university settings confers the same advantage on course performance or influences study methods employed in the course. In other words, students who have had experience in anatomical coursework may have developed effective study approaches before taking a more advanced anatomy course. The purpose of this study is to assess the effect of previous human anatomy coursework on lecture and lab exam performance and study habits in an undergraduate human anatomy course at a large state university in Tennessee for the 2018–2019 and 2019–2020 academic years (N = 442).

The course enrollment consists of nursing, kinesiology, or pre‐professional undergraduate majors, of which more than half (53%) are sophomores, and the remainder juniors (17.5%) and seniors (29.5%). We collected data from students at midterm through an anonymous survey. Students self‐reported their performance on assessments for the first major examination in the course, which has a written lecture component and a practical lab component. The survey also included questions about previous anatomy coursework, prior enrollment in the current course, study habits through a range of modalities, and use of extracurricular resources. We performed nonparametric Kruskal‐Wallis tests to examine the relationship between prior anatomy experience and performance on initial lecture and lab assessments, as well as study methods. Results indicate that no significant difference in first lecture or lab assessment performance is associated with prior anatomy coursework, regardless of prior course setting (high school versus college). Additionally, prior coursework in anatomy is not associated with a significant difference in when students began studying for their first lecture/lab assessments. There is a trend for students with prior coursework in anatomy to perform slightly better on assessments than those who had not. In addition, preliminary analysis of study modalities shows that students with prior anatomical experience do not engage in study methods different from those who have not taken an anatomy course before.
Combination Effect of Tofacitinib and Berberine for the Treatment of Dextran Sulfate Sodium‐Induced Chronic Colitis in C57BL/6 Mice
Authors: Li Yanhong , Tang Huazheng
Published: April 2020

Background and Aims

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon with a relapsing–remitting pattern that belongs to autoimmune disease (AID). The JAK inhibitor tofacitinib is a newly (2018.05) FDA approved drug for UC which is a drug previously used to treat another AID, rheumatoid arthritis. A higher dose of tofacitinib has been associated with a safety warning for increased lipid levels as well as serious infections and malignancy. Berberine has proved to be a promising drug candidate for the treatment of UC and shows activity in hyperlipidemia, bacteria; infections and cancer. We hypothesized that a balance between clinical benefit and side effects might be attained by using a combination of tofacitinib and berberine‐‐with reduced doses of both drugs. We first investigated the preclinical activity of reduced doses of tofacitinib plus berberine in experimental colitis in the mouse.

Methods

Tofacitinib (30 mg/kg/day), berberine (40 mg/kg/day) and reduced doses of tofacitinib (15 mg/kg/day) plus berberine (20 mg/kg/day) were administered orally for 14 and 30 days to C57BL/6 mice with colitis induced by two schedules of three cycles of 2% dextran sulfate sodium (DSS). Disease severity, colon length, colon and spleen swelling and cytokine expression in colon tissue as well as histology of major organs were examined.

Results

The results showed that compared to the two drugs alone, half reduced doses of tofacitinib plus berberine showed a comparable effect in ameliorating DSS‐induced colitis severity and colon shortening. Co‐treatment showed a greater antagonism of DSS induced colon and spleen swelling when compared to either drug alone. Similar effects were observed in cytokine levels in the colon. In addition, no potential toxicity to major organs was observed with this combination therapy.

Conclusions

Our studies provide a preclinical rationale for the combination of reduced dosage tofacitinib and berberine as a possible clinical therapeutic strategy for the treatment of UC.

Support or Funding Information

This work was funded by the Research Foundation of South China University of Technology (x2yxD2182200), the Natural Science Foundation of Guangdong Province (2018A030310388), Guangdong Medical Science Foundation (A2018363), and China Scholarship Council (Grant No. CSC201806155103).
Localization of NUCB2/Nesfatin‐3/Nesfatin‐1 in Normal and Inflamed Human and Mouse Lungs, and Human Neutrophils
Authors: Singh Baljit , Hui Jasmine , Singh Atul K.
Published: April 2020

Nesfatin‐1 regulates hunger and fat storage and is produced in the hypothalamus of mammals. Nucleobindin2 (NUCB2)/nesfatin‐1 protein expression in human plasma positively correlates with expression of pro‐inflammatory cytokines in patients with chronic obstructive pulmonary disease (COPD) suggesting its potential role in lung inflammation. However, we still don’t precisely know the expression of NUCB2/nesfatin‐1 in lungs and neutrophils. In this study, the expression of NUCB2/nesfatin‐3/nesfatin‐1 in normal and inflamed human and mouse lungs and neutrophils were examined with light and electron microscopic immunocytochemistry. Results from light microscopy showed that the localization of NUCB2/nesfatin‐1 in the pulmonary epithelium, alveolar septa, vascular endothelium and various immune cells does not change with inflammatory status. Electron microscopy revealed constitutive localization within the nucleus and cytoplasm of the aforementioned cells. Further, NUCB2/nesfatin‐1 accumulated within 0.5μm of the plasma membrane in human neutrophils following 90mins of 1ng/mL LPS stimulation. NUCB2/nesfatin‐3 was also found to localize in euchromatic portions of neutrophilic nuclei at 5 times the mean concentration compared to heterochromatin. Finally, our results indicate that NUCB2/nesfatin‐3 is predominantly cytoplasmic as it localizes at 2 times the concentration in neutrophilic cytoplasm compared to nucleus. Our study is the first to detail the localization of NUCB2/nesfatin‐1/nesfatin‐3 in lungs and neutrophils, and also implicate their potential role in transcriptional regulation.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada
The Plasminogen Receptor, Plg‐RKT, Regulates Inflammation and Fibrinolysis During Wound Healing
Authors: Miles Lindsey A. , Ny Lina , Holmberg Sandra , Baik Nagyung A. , Bäckman Assar , Broden Jessica , Parmer Robert J. , Wilczynska Malgorzata , Ny Tor
Published: April 2020

Plasminogen is a key regulatory protein that functions to both promote fibrinolysis and to potentiate the inflammatory response during wound healing. Here we have tested the hypothesis that the novel plasminogen receptor, Plg‐RKT, regulates distinct steps in the wound healing process.

Method

Full‐thickness standardized burn wounds (1 cm in diameter) were induced in mice and the wound area was quantified at different time points. Wound tissue was subjected to qPCR and ELISA for cytokine expression and immunohistochemistry and Western blotting for fibrin deposition and leukocyte recruitment.

Results

From day 9 after injury, healing in Plg‐RKT−/− mice was significantly delayed compared with healing in Plg‐RKT+/+ littermates. Expression of cytokines, IL‐1β, CCL‐20, TNFα, and IL‐10 was dysregulated in Plg‐RKT−/− tissue although no genotype‐dependent differences in recruitment of macrophages or neutrophils to the wound area were detected. Consistent with dysregulated cytokine expression, a significant delay in wound healing during the proliferation phase (days 9 and 10) was observed in wound tissue from mPlg‐RKT−/− mice in which Plg‐RKT was specifically deleted in myeloid cells. Regarding fibrinolysis, fibrin clearance was significantly impaired in Plg‐RKT−/− wound tissue. Genetic reduction of fibrinogen levels of Plg‐RKT−/− mice to 50% completely abrogated the effect of Plg‐RKT deletion on healing of burn wounds and, remarkably, effects of Plg‐RKT deletion on cytokine expression were modulated by the reduction in fibrinogen levels. In conclusion, Plg‐RKT plays a key role in wound healing by regulating both fibrinolysis and the inflammatory response and fibrin plays a role in regulating Plg‐RKT function on myeloid cells.

Support or Funding Information

United States National Institutes of Health grants HL 081046 to LAM and HL 149511 to LAM and RJP and Merit Review Award #5I01BX002026 from the U.S. Department of Veterans Affairs to RJP.
Role of PLA2R1 and sPLA2 on Drug Release and Uptake of Liposome Nanoparticles in Prostate Cancer
Authors: Alnaim Ahmed Saad , Arnold Robert , Eggert Matthew , Nie Ben , Jasper Shanese , Davis Joshua , Panizzi Peter , Cummings Brian , Quach Nhat
Published: April 2020

Exploiting differences in tumor pathophysiology can enhance the delivery and antitumor activity of chemotherapeutics encapsulated in drug carriers. Long‐circulating nanoparticulate drug carriers, such as pegylated‐sterically‐stabilized liposomes (SSL), can stably entrap drug, alter drug disposition, improve antitumor activity and minimize toxicity. However, control of their drug‐release kinetics has limited their clinical potential. Secretory phospholipases A2 (sPLA2) are over expressed and excreted in a variety of tumors, e.g., up to 22‐fold in prostate. These enzymes degrade phospholipids preferentially and have been hypothesized as targets to control drug release from lipid‐based nanoparticles, such as liposomes. While they have shown activity in preclinical models, the clinical performance of these formulations has been limited. The goal of these studies was to gain insights into how sPLA2‐targeted liposome formulation (SPRL) alter drug release and uptake, specifically examining the role of sPLA2 and its membrane receptor (PLA2R1). Studies were performed using a metastatic‐derived human prostate adenocarcinoma cells (PC‐3) and a PLA2R1 knock‐down variant (PC‐3‐PLA2R‐KD) while varying supplementation of sPLA2 enzyme isoforms (IIA, X, V). Liposomes (SSL & SPRL) were made as described previously and included DiR, a non‐exchangeable near‐infrared fluorescent dye. In vitro uptake and deposition of various formulations was determined using flow cytometry and fluorescence microscopy by measuring DiR and doxorubicin (fluorescent anticancer agent) entrapped within some of the formulations. Biodistribution and circulation times were determined non‐invasively on an IVIS Lumina XRMS and iThera Multispectral Optoacoustic Tomography (MSOT) systems in Athymic mouse xenograft models of prostate cancer. Liposomes formulations of SPRL and SSL were prepared at size of 120±1 and 118±2 nm. Circulation half‐life and tumor deposition was similar for both formulations, suggesting the enhanced pharmacological activity is related to release/uptake after drug extravasation. In‐vitro studies showed that PC‐3‐PLA2R‐KD cells had greater DiR fluorescence of both formulations than wild PC‐3 after 48 hr. Moreover, SPRL had greater uptake than SSL formulation (p<0.05) beyond 24 hr. In‐vivo studies showed greater uptake of SSL and SPRL in the PC‐3‐PLA2R‐KD tumors. Overall these data suggest that PLA2R1 plays an important role in the performance of SSL and SPRL formulations. Further studies are needed to gain insights into the clinical significance and to determine if presence of PLA2R1 can be used to identify specific patient populations that would best respond to treatment with SPRL.

Support or Funding Information

NIH/NIBIB 1R01EB016100‐01(RDA/BSC), 1R21EB008153 (RDA/BSC), and AU Research Initiative in Cancer “AURIC”.
GPR139 Signals Through Gq/11 to Oppose Mu Opioid Receptor Signaling
Authors: Stoveken Hannah M. , Zucca Stefano , Masuho Ikuo , Wang Dandan , Dao Maria , Grill Brock , Martemyanov Kirill A.
Published: April 2020

Signaling through G protein coupled receptors (GPCRs) is highly regulated and shaped not only by how and when a GPCR engages its ligand, but also through complex and varied protein interactions at the level of the receptor and at downstream effectors. Opioid receptors are an excellent example of a GPCR system that is finely regulated in the central nervous system. The mu opioid receptor is the primary receptor that modulates acute pain responses and has been heavily targeted therapeutically for its anti‐nociceptive properties. The duration and extent of endogenous MOR signaling is modulated by available endogenous opioid peptides, by β‐arrrestin recruitment and subsequent receptor desensitization, by regulator of G protein signaling (RGS) control of Gαo activation, and by interaction with other GPCRs that may positively or negatively alter signaling. Using an unbiased forward genetic screen in MOR‐expressing c. elegans, we uncovered another possible regulator of MOR signaling: the orphan GPCR GPR139. Studies using GPR139 knockout mice demonstrate hypersensitivity to opioid analgesics across multiple measures of antinociception. However, the exact molecular mechanism(s) of how GPR139 activation and signaling can modulate MOR downstream signaling is unknown. We ran a battery of in vitro signaling assays to determine how GPR139 signals in cells and how that could negatively modulate MOR signaling. GPR139 G protein coupling was tested using every G alpha protein and was found to couple to both Gq/11 and Gi/o family of G proteins. We examined the effect of GPR139 on multiple parameters of MOR signaling and trafficking and found that GPR139‐Gq signals in an opposing manner to MOR‐Go signaling at multiple downstream effectors in vitro. Further, MOR‐mediated changes in habenular neuron firing are blunted by dynamic GPR139 activation and can be rescued by Gαq inhibition. Overall, this research examines the molecular underpinnings of GPR139 negative regulation of MOR, explores mechanisms of GPCR cross‐regulation, and evaluates the possibility of therapeutically targeting GPR139 to increase the safety and efficacy of opioids.

Support or Funding Information

National Institute on Drug Abuse (NIDA) NRSA F32DA047771 to HMSNational Institute on Drug Abuse (NIDA) DA042746 to KAM
Profiling of Inflammatory Biomarkers and Coagulation Factors in End‐Stage Renal Disease
Authors: Bontekoe Emily , Grewal Arjun , Bansal Vinod , Tahar Meriam Ben Hadj , Hoppensteadt Debra , Fareed Jawed
Published: April 2020

Background

Early diagnosis of chronic kidney disease (CKD) is necessary in preventing complications that develop into kidney failure and vascular complications. Currently, the detection of early stages of CKD is lacking predictive values, delaying the application of specific diagnostics and therapeutic interventions. End‐stage renal disease (ESRD) patients are at risk for cardiovascular complications and vascular thrombosis, exhibiting numerous coagulation and fibrinolytic abnormalities.

Objectives

Profiling inflammatory biomarkers and coagulation factors in ESRD patients may allow early onset detection and severity of kidney damage, furthering the understanding of the pathogenesis of this disease.

Materials & Methods

Citrated blood samples were collected from 95 confirmed ESRD patients undergoing maintenance hemodialysis under an IRB approved protocol. Biomarkers of inflammation and coagulation, including PAI‐1, TAFI, CRP, MMP‐9, MP, D‐Dimer, Factor VII, IX, X, and XIII were measured using commercially available ELISA methods. Normal controls were comprised of commercially available male and female citrated plasma (George King Biomedical). All results were compiled as mean ± SEM, and percent change in comparison to normal human plasma.

Results

Biomarkers including CRP (7ug/ml ± 0.6, 183%), MMP‐9 (88ng/ml ±8, 58%) D‐Dimer (1059ng/ml±138), Factor IX (124%±3, 23%), and Factor XIII (104%±1, 27%) showed significant increase when compared to normal plasma. Elevated CRP indicates severe inflammatory response and elevated D‐Dimer indicates endogenous fibrin formation. MP and PAI‐1 suggest endothelial dysfunction.

Discussion

These results indicate that the upregulation of inflammatory responses and hemostatic defects contribute to the multifactorial pathophysiology of ESRD. Profiling of these biomarkers may be helpful in the prognostic management of these patients.
CDK5 promotes ventricular arrhythmogenesis through phosphorylation of N‐type calcium channels in cardiac sympathetic postganglionic neurons
Authors: Zhang Dongze , Tu Huiyin , Hu Wenfeng , Wadman Michael C. , Li Yulong
Published: April 2020

Cardiac sympathetic overactivation is an important trigger of fatal ventricular arrhythmias in patients with chronic heart failure (CHF). Our previous study found that increased N‐type calcium (Cav2.2) currents in cardiac sympathetic postganglionic (CSP) neurons contributes to cardiac sympathetic overactivation and ventricular arrhythmogenesis in CHF rats. Cyclin‐dependent kinase 5 (CDK5), a proline‐directed serine/threonine kinase, has been reported to play an important role in regulation of calcium influx and neurotransmitter release through phosphorylation of Cav2.2α subunit. Our current study aims to investigate whether CDK5 induces cardiac sympathetic overactivation and increases the susceptibility to ventricular arrhythmias via phosphorylation of Cav2.2α in CSP neurons. Lentiviral‐CDK5 cDNA (1×107 TU/ml, 2μl) was in vivo transfected into bilateral stellate ganglia (SG) in sham rats. Nontransfected sham rats served as controls. Final experiments were performed at 9 days after gene transfection. Western blot data demonstrated that gene transfection with lentiviral‐CDK5 cDNA into SGs significantly increased protein expression of CDK5 in CSP neurons in sham rats, compared with nontransfected sham rats. CDK5 cDNA transfection in SGs increased phosphorylation level of Cav 2.2α in CSP neurons from sham rat. The power spectral analysis of heart rate variability in conscious rats demonstrated that overexpression of CDK5 in CSP neurons increased low frequency power and ratio of low frequency to high frequency (index of cardiac sympathetic activation). Additionally, transfection of CDK5 cDNA into CSP neurons markedly increased cardiac sympathetic nerve activity in anesthetized sham rats (45.3±6.4% in sham+CDK5 group vs. 29.2±2.1% in sham group). ECG data obtained from 24‐hour continuous telemetry ECG recording revealed that overexpression of CDK5 in CSP neurons induced heterogeneity of ventricular electrical activities in conscious sham rats, as evidenced by CDK5 cDNA‐elongated ventricular arrhythmia‐related ECG markers including QT interval, QT dispersion, and T‐peak to T‐end interval. More importantly, transfection with CDK5 cDNA into CSP neurons significantly increased the susceptibility to ventricular arrhythmias (arrhythmia score was 1.7±0.9 in sham+CDK5 group vs. 0 in sham group) in anesthetized sham rats. Based on above data, we conclude that CDK5 induces cardiac sympathetic overactivation via phosphorylation of Cav 2.2‐α subunits in CSP neurons, and further increases the susceptibility of ventricular arrhythmias in sham rats.

Support or Funding Information

This study was supported by National Heart, Lung, and Blood Institute Grant (R01HL137832‐01 to Y.‐L. Li)
Proteomic and Phosphoproteomic Analysis of Skeletal Muscle During Hindlimb Immobilization
Authors: Lin Kuan-Hung , Blanco Rocky , Wilson Gary M. , Steinert Nate D. , Coon Joshua J. , Hornberger Troy A.
Published: April 2020

The maintenance of skeletal muscle mass is required for health and issues associated with the quality of life. It is known that immobilization leads to the loss of muscle mass, and a decrease in protein synthesis is one of the primary mechanisms that drive this response. Moreover, previous studies have shown that protein synthesis is largely regulated by changes in the phosphorylation state of the various factors that can control protein synthesis (e.g., initiation and elongation factors). In this study, we took advantage of the powerful capabilities of mass spectrometry (MS) to identify the proteomic and phosphoproteomic alterations that occur in response to immobilization. In total, we were able to identify and quantify alterations in 15,020 unique phosphorylation sites and 4,646 proteins. To our surprise, the rate of protein synthesis decreased within 6 hours of immobilization, but no phosphorylation sites were significantly altered at this time. At later time points (i.e., 24 and 72 hours), the rate of protein synthesis further decreased, but again this was not associated with major changes in protein phosphorylation. To verify the proteomic and phosphoproteomic results, we performed western blot analysis to assess the phosphorylation state of various proteins that have been implicated in the regulation of protein synthesis. The results from the western blot analysis were highly congruent with the MS results. Hence, when taken together, our results strongly suggest that alterations in protein phosphorylation do not play a major role in immobilization‐induced decrease in protein synthesis and muscle mass.

Support or Funding Information

National Institutes for Health grant 1R01AR057347 to T.A.H.