bluuebunny/crossref_metadata_2025 huggingface dataset:
Zinc Deficiency‐Induced NFkB Activation Promotes Hypertension by Driving Renal Na+ Reabsorption
Authors: Williams Clintoria R. , Schindele Dylan S. , Murta Cindellynn K. , Wenegieme Tara-Yesomi , Waite Aston , Sonner Martha J.
Published: April 2020

Background

Zinc deficiency (ZnD) is a common comorbidity with chronic disorders including diabetes and kidney disease. Patients with these diseases also have a higher incidence of hypertension. Recently, we reported that a zinc deficient diet is sufficient to induce hypertension in mice. Changes in blood pressure were accompanied by increased Na+ reabsorption via the renal sodium chloride cotransporter (NCC). Although our novel results indicate that zinc plays a critical role in NCC regulation and its downstream effects on blood pressure, the specific mechanisms involved are unknown.

Hypothesis

Since nuclear factor‐kB (NFkB) is a key transcription factor implicated in ZnD‐mediated effects, we tested the hypothesis that ZnD stimulates renal NFκB activation which drives NCC upregulation and thereby promotes hypertension.

Experimental Design

Adult, male C57BL/6 mice were fed a zinc adequate (ZnA; 50ppm) or a ZnD (1ppm) diet for 6 weeks. To examine the role of NFkB in ZnD‐induced hypertension, mice were switched from a ZnA‐diet to a ZnD‐diet for 5 weeks. After, ZnD mice were administered caffeic acid phenethyl ester (CAPE), an NFkB inhibitor, for 1 week. In mice, NCC expression (qRT‐PCR) and blood pressure (tail‐cuff) were measured. To further investigate the role of NFkB in NCC expression (qRT‐PCR and Western blot), mouse distal convoluted tubule (mDCT) cells were treated with TPEN (a zinc chelator) or vehicle (DMSO) ± CAPE.

Results

Compared to ZnA mice, NCC mRNA abundance was increased in ZnD mice (7.625 ± 1.747 vs 1.0 ± 0.127) and was accompanied by enhanced renal NFkB nuclear localization. In mDCT cells, TPEN‐induced ZnD stimulated NCC mRNA expression (3.152 ± 0.966 vs 1.0 ± 0.174) and NFkB upregulation (1.573 ± 0.117 vs 1.0 ± 0). However, NFkB inhibition by CAPE prevented TPEN‐induced NCC mRNA (0.636 ± 0.178 vs 3.152 ± 0.966) and protein (1.039 ± 0.018 vs 2.335 ± 0.062) expression in mDCT cells. Finally, CAPE administration lowered blood pressure (154.5 ± 5.42 vs 138.1 ± 1.97 mm Hg) in ZnD mice.

These results demonstrate that 1) renal NFkB is a Zn2+‐regulated transcription factor, 2) NFkB is involved in NCC regulation and 3) NFkB mediates ZnD‐induced blood pressure increases.

Significance

Taken together, these data indicate that ZnD stimulates NFkB activation thereby driving NCC upregulation and hypertension.

Support or Funding Information

AHA‐16SDG27080009 and NIH‐R21DK119879
Mechanism of Acrolein Toxicity:The Effect on Epigenetic Changes
Authors: Nwaiwu Uchechi Grace , Yousefipour Zivar , Ranganna Kasturi , Joseph Mathew
Published: April 2020

Acrolein, an alpha‐beta unsaturated aldehyde and a very reactive toxic compound, is released into the environment from different sources such as combustion of fossil fuel, cigarette smoke, overheating of frying oil, as well as endogenously being produced in body via lipid peroxidation. As a pervasive environmental pollutant, acrolein poses a serious environmental health threat acknowledged by investigators, health, and environmental government agencies.

Epigenetics is the modifications in gene expression that do not alter the DNA sequence of a gene. This alteration could occur naturally or by factors such as age, environmental exposure, individual lifestyle, and disease condition. Phosphorylation, Methylations and acetylation are the most common epigenetic changes. N‐ Acetyl cysteine (NAC) is an antioxidant involved in the production of cellular glutathione.

The aim of the study was to determine the effect of acrolein on histone phosphorylation, acetylation and methylation in vascular smooth muscle cells (VSMCs) and to establish the beneficial effect of NAC in prevention of acrolein toxicity through blockage of histones modification.

VSMCs were treated with 3μg/ml acrolein for 6 hours in the presence and absence of 0.2 mM NAC. At the end of the treatment, cytotoxicity of acrolein was measured using tetrazolium‐calorimetric assay. Additionally, free radical generation was measured by fluorometric methods, histone H3 modification by ELISA kit, and protein expression by western blotting.

Results indicated that six‐hour exposure of VSMC to 3μg/ml of acrolein resulted in 99% decrease in cell viability and addition of 0.2Mm of NAC provided complete protection of VSMCs from toxicity caused by acrolein. In the absence of NAC, acrolein increased level of reactive oxygen species (ROS) generation by 85%. Addition of NAC abolished high level of ROS by 75%.

ELISA analysis showed acrolein‐induced modification of histones 3 (H3) at different lysine and serine residues including SER‐10P and SER‐28P, K4ME, K9ME, K27ME, K79ME, K9AC, K14AC, K18AC. Western blot analysis of two of these residues indicated 45% increase in Ser10P1, 59% increase in Ser28p1, and addition of NAC decreased these modifications by 86% and 76% respectively. Additionally, we observed a 66% increase in the expression of methylated H3K9 and 66% in acetylated H3k9 protein, which was attenuated by addition of NAC by 71% and 65% respectively.

In conclusion, this study revealed that exposure of VSMCs to acrolein causes modification of H3 histones at different residues and NAC is effective in inhibiting the effects of acrolein.

Support or Funding Information

NIH SC3GM103746‐04
Subcellular Drug Targeting Illuminates Local Action of Polo‐like kinase 1 and Aurora A During Mitosis
Authors: Bucko Paula J. , Lombard Chloe K. , Rathbun Lindsay , Bhat Akansha , Garcia Irvin , Wordeman Linda , Smith F Donelson , Maly Dustin J. , Hehnly Heidi , Scott John D.
Published: April 2020

Fundamental cellular processes such as cell division, migration, metabolism, and growth rely on the precise and timely regulation of signaling proteins. Yet how individual signal transduction events at specific microenvironments drive complex cellular processes remains elusive. During mitosis, two protein kinases, polo‐like kinase 1 (Plk1) and Aurora A (AurA) share several overlapping functions. Current approaches in which small‐molecule kinase inhibitor drugs are used to ascribe function to Plk1 and AurA provide important insight into the global roles of these proteins inside the cell. Unfortunately, the indiscriminate distribution of such traditional inhibitor drugs limits our ability to resolve how these mitotic enzymes function at distinct organelles. Here we exploit the SNAP‐tag system to create genetically encoded platforms that restrain kinase inhibitor drugs at defined subcellular locations.
Local
Kinase
Inhibition (LoKI) allows us to ascribe organelle‐specific functions to broad‐specificity kinases. Using a combination of biochemical approaches, quantitative imaging, and live‐cell microscopy we uncover that local targeting of Plk1 and AurA kinase inhibitors to centrosomes disrupts substrate phosphorylation, spindle formation, and mitotic duration more profoundly than global drug distribution in human cells. Furthermore, we show that zebrafish embryos treated with centrosome‐targeted Plk1 inhibitors have more microtubule abnormalities than those treated with a non‐localized inhibitor. Finally, we expand our approach by targeting drugs to the kinetochore and find that inhibition of kinetochore‐associated pools of AurA blocks phosphorylation of microtubule‐kinetochore components. Future work will employ LoKI to investigate how downstream substrates of Plk1 and AurA function at centrosomes and kinetochore to coordinate mitotic signaling events.
New nonintrusive method of predicting adult stature from the age of 13 years using chronological age and maturity. Validation in adolescent basketball players
Authors: Duvallet Alain Louis , Pineau Jean-Claude , Duvallet Emilie , Lhuissier François , Beaudry Michele
Published: April 2020

The purpose of this study was to define a new method to estimate the adult height of an individual from data recorded during a single and nonintrusive childhood examination. The data required are maturity stage and stature in a standing position. Specific growth curves were obtained from longitudinal data collected from 125 sedentary boys aged from 12 to 17.5 years, during 5 consecutive years. The sample comprise of 1234 measurements.

The puberty stages were based on facial, axillary hair growth and changing voice with four stages: stage 1 (ST1, below the puberty), stage 2 (ST2, prepubescent), stage 3 (ST3, para pubescent), and stage 4 (ST4, pubescent). They were classified as early, standard, or late maturation.

Between the different maturation groups, significant differences were found for the age of peak height velocity (APHV). From 160 to 164 months, we found a connection between the pubertal stages and the APHV without possible intersection. Significant differences were observed between APHV for each maturation group, which enable to model average curves of the stature for each puberty group.

To validate the new model, we predicted young basketball players’ adult stature. First, data were collected (height and stage of puberty) a single time for age between 160 and 164 months. From our models of sedentary reference subjects we estimated the adult stature. In a second time, these estimations were compared to the real measured stature at 18 years old (216 months).

For each basketball player we calculated the deviation from the average value obtained on the first population expressed as standard deviation (Z‐score).

The relation between the predicted stature and the adult stature for overall basketball population shows a correlation with r = 0.98 and SEE = 1.65 cm. The predictions lie within +/− 0.3cm, 98% of the time.

This study shows that the knowledge of biological maturation (peak of height velocity) and maturity (delayed, standard or advanced puberty) make possible to develop new stature growth curves, and to predict adult stature.

This technique is a new valid nonintrusive and accurate method, which permits to predict adult stature. It was validated with a population of basketball players and constitutes an advance in the detection and the orientation of future basketball players.

Support or Funding Information

Michele Beaudry
Reliability of Skin Blood Flow Measurement with Multiple Laser Doppler Probes
Authors: Cui Jian , Kunselman Allen R. , Herr Michael D. , Pai Samuel , Blaha Cheryl , Luck Jonathan Carter , Sinoway Lawrence I.
Published: April 2020

Skin blood flow (SkBF) measurements with laser Doppler probe provide a continuous real‐time index for local microcirculatory blood flow under the probe. However, because the area detected by a single probe is very small, it is known that SkBF reading may vary significantly if the position of the probe is moved slightly. The possible low reliability after replacing the probe limits the application of this measurement in some experiments (e.g. prior and after outdoor exercise). In this study, we tested the hypothesis that using multiple laser Doppler probes could increase the reliability of the measurements. SkBF, local skin temperature (Tsk) and blood pressure were measured with 3 repeated trials in 11 subjects (6 M, 5 F, 42 ± 5 yrs) in a temperature controlled room. Seven laser Doppler probes were placed in a custom‐made holder (round shape, diameter 45 mm). Trial 1: after the probe holder was placed on a forearm, the position was marked. After ~5 min rest, SkBF and Tsk (under the holder) were recorded for 3 min. Then, the probe holder was removed from the forearm and placed on the side calf of a leg to measure leg SkBF. After dis‐instrumentation, subjects were released from the testing table for 10–15 min. Trial 2 and 3: the probe holder was carefully replaced as close as possible on the same positions according to the markers on the skin. The measurements on forearm and then leg as well as the intervals between the trials were repeated. In this Test‐Retest Reliability study, two‐way mixed‐effects models with absolute agreement were used to compute the intraclass correlation coefficient (ICC). There was no significant difference in blood pressure and Tsk between the trials. With the measurements from each single probe, the lowest ICCs were 0.68 (95% Confidence Interval: 0.35, 0.89) for SkBF and 0.76 (0.48, 0.92) for cutaneous vascular conductance (CVC) at leg. With the averaged values from 7 probes, the ICCs were 0.86 (0.62, 0.96) for SkBF and 0.91 (0.74, 0.97) for CVC at leg, and 0.95 (0.87, 0.99) for SkBF and 0.96 (0.88, 0.99) for CVC at forearm. Moreover, with the averaged values from 4 contiguous probes, the ICCs were greater than 0.84. These data suggest that SkBF measurement with multiple laser Doppler probes in a custom‐made holder has high reliability after replacing the probes on the same subjects.

Support or Funding Information

Supported by NIH MPI R01 HL141198 (Li and Cui) and UL1 TR002014 (Sinoway)
DNA Replication Checkpoint Activates Respiration in Budding Yeast
Authors: Nagar Shreya , Kaur Pritpal , Bhagwat Madhura , Jassar Salony , Vancura Ales
Published: April 2020

In addition to DNA damage checkpoint (DDC), eukaryotic cells have also DNA replication checkpoint (DRC) that is distinct from the DDC and specifically signals slowly progressing or arrested replication forks. We have showed previously that DDC activates respiration to increase ATP production and to elevate dNTP levels, which are required for efficient DNA repair and cell survival upon DNA damage. The underlying mechanism involves DNA damage‐induced downregulation of histone expression and altered chromatin structure, leading to increased transcription of genes encoding enzymes of tricarboxylic acid cycle, electron transport chain, and oxidative phosphorylation. Here we show that similarly to DDC, activation of DRC also induces respiration by a Mec1p‐ and Rad53p‐ dependent mechanism. However, unlike DDC, DRC induces respiration by a mechanism that is not dependent on histone gene expression and chromatin structure.

Support or Funding Information

NIH GM120710
Regulation of Histone Gene Expression during DNA Damage Response in Budding Yeast
Authors: Bhagwat Madhura , Nagar Shreya , Kaur Pritpal , Vancura Ales
Published: April 2020

The DNA damage response (DDR), a highly conserved process, senses and repairs damaged DNA. We have showed previously that DDR activates respiration to increase ATP production and to elevate dNTP levels, which are required for efficient DNA repair and cell survival upon DNA damage. The underlying mechanism involves DNA damage‐induced downregulation of histone expression and altered chromatin structure, leading to increased transcription of genes encoding enzymes of tricarboxylic acid cycle, electron transport chain, and oxidative phosphorylation. Here we show that the downregulation of histone gene expression during DDR requires Rad53p and occurs at the level of transcription and not at the level histone mRNA decay. The corresponding mechanism involves DDR‐mediated inhibition of assembly of the preinitiation complex and decreased recruitment of Spt10p and Spt21p to the promoters of histone genes.

Support or Funding Information

NIH GM120710
Correlation of MCAT scores and preparative study material to USMLE Step I performance
Authors: Seal Zachary Alvis , Koek Wouter , Sharma Ramaswamy
Published: April 2020

The Medical College Admission Test (MCAT) is a well‐utilized metric for stratifying students for medical school admissions. The United States Medical Licensing Examination (USMLE) is a three‐step examination that assesses student knowledge of gross anatomy, biochemistry, biostatistics, genetics, and other basic science disciplines required for practicing medicine in the United States. The MCAT has been shown to correlate positively with student success on Step 1. However, in 2015, a new MCAT format was released and its correlation with Step 1 remains to be analyzed. Additionally, preparation for Step 1 typically involves purchasing and perusing practice tests from the National Board of Medical Examiners (NBME®) and UWorld®; however, their predictive value to performance on Step 1 remains to be ascertained, especially with the release of five new NBME® practice tests. We hypothesized that the new MCAT scores correlated positively with Step 1 scores and that the new NBME® practice tests were more predictive of performance on Step 1 as compared to old NBME® tests. An anonymous survey was posted on www.reddit.com requesting users to submit their scores on Step 1, practice tests and MCAT along with examination dates. Regression analysis indicated positive correlation of MCAT with Step 1 scores (P= 1.236E‐15; R2=0.2047; n=282). While new NBME® practice tests were more predictive of Step 1 scores than old NBME® tests, UWorld® SA2 scores were most predictive (P=3.752E‐93; R2=0.6803; n=370). Taking more than six practice tests significantly enhanced Step 1 scores (P=0.040). Data from this study can be used to create a mathematical framework using MCAT and practice test scores for accurate prediction of Step 1 scores.
Serum lead and cadmium exposure is an independent risk factor for acute ischemic stroke in a south Taiwan population
Authors: Chen Hsin-Hung , Yen Cheng-Chung , Hsu Yi-Ting , Cheng Pei-Wen , Tseng Ching-Jiunn , Lin Ching-Huang
Published: April 2020

Background

Acute ischemic stroke (AIS) is among the major causes of disability and death worldwide. It is caused by thrombotic or embolic occlusion of a cerebral artery and has a higher prevalence than hemorrhagic stroke. Environmental exposure to heavy metals has been associated with AIS risk; however, this association is yet to be well established. We aimed to assess the effect of accumulation of heavy metals (e.g. arsenic, cadmium, lead, and mercury) in patients with AIS.

Methods

We enrolled 33 pairs of newly diagnosed patients with AIS. We used inductively coupled plasma mass spectrometry to measure heavy metal levels. We used Pearson’s correlation coefficient to assess the association between heavy metal levels and the variables. Between‐group differences in the heavy metal levels and variables were analyzed using an independent t‐test.

Results

Serum cadmium levels were significantly higher in patients with AIS who were smokers (mean ± SD: 1.0 ± 0.5 mg/L versus 0.6 ± 0.2 mg/L; P < 0.003). Similarly, serum lead levels were significantly higher in smokers (mean ± SD: 25.3 ± 8.5 mg/L versus 17.5 ± 6.3 mg/L; P < 0.006). Further, serum cadmium levels were significantly higher in alcohol drinking patients with AIS (mean ± SD: 1.2 ± 0.6 mg/L versus 0.6 ± 0.3 mg/L; P < 0.003). Similarly, serum lead levels were significantly higher in alcohol drinkers (mean ± SD: 29.5 ± 8.6 mg/L versus 18.5 ± 6.1 mg/L; P < 0.003).

Conclusions

Serum cadmium and lead levels were associated with the incidence of AIS both independently and in association with smoking and drinking. This supports the hypothesis that smoking and drinking increases cadmium and lead exposure, and therefore increases the AIS risk.

Support or Funding Information

107‐2320‐B‐075B‐002‐MY2、107‐2320‐B‐075B‐001
Voluntary exercise as a preventive strategy against aircraft noise induced cardio/cerebrovascular damage
Authors: Kroeller-Schoen Swenja , Kvandova Miroslava , Schmal Isabella , Kalinovic Sanela , Stamm Paul , Frenis Katelyn , Daiber Andreas , Oelze Matthias , Schulz Eberhard , Jansen Thomas , Munzel Thomas
Published: April 2020

Background

Several experimental studies found transportation noise to be associated with oxidative stress‐induced vascular damage, mediated by activation of the NADPH oxidase, uncoupling of endothelial nitric oxide synthase, and vascular infiltration with inflammatory cells. Furthermore, we could show that the AMP‐dependent‐Kinase (AMPK) is involved in the signaling processes leading to exercise‐mediated vascular protection. In the present study, we investigated the activation of AMPK during voluntary exercise as a preventive strategy.

Methods and Results

Wild type (WT) mice were exposed to aircraft noise (maximum sound level of 85 dB(A), average sound pressure level of 72 dB(A) for 4 days after 6 weeks of voluntary exercise. We could show that 4 days noise exposure lead to a significant endothelial dysfunction accompanied by and significant increase in oxidative stress. The voluntary exercise training for 6 weeks resulted in a marked improvement of noise induced endothelium dysfunction and activation of ROS sources (NADPH oxidases). The beneficial effects of exercise training in the vasculature are accompanied by improvement of cerebral oxidative stress due to NADPH oxidases (NOX‐1, NOX‐2) and cerebral inflammatory responses (VCAM‐1, PAI‐1, COX‐2, MCP‐1). Transportation noise is disrupting gene expression patterns of circadian rhythm but we could show that exercise training is able to reestablish aortic circadian clock gene expression in noise exposed wild type mice.

Conclusion

We here present novel data on exercise training as a protective strategy against negative effects of aircraft noise exposure by conferring AMPK activation. Our data suggest that exercise training might be an attractive mitigation strategy to protect the general population from noise‐induced cardiovascular damage. Especially patients at high cardiovascular risk could benefit from supervised exercise training to prevent additive cardio/cerebrovascular damage in response to transportation noise exposure.

Support or Funding Information

German Research Foundation (Deutsche Forschungsgemeinschaft) DFG
Enteromorpha prolifera Extract Protects against Scopolamine‐induced Memory Impairment in Mice
Authors: KIM MEE REE , Baek Seong Yeon , Li FuYi
Published: April 2020

Enteromorpha prolifera (EP) contains various nutrients and phytochemicals that have been reported to play role in antioxidant activity, hypolipidemic and immunomodulatory. However, the neuroprotective effect of EP has not been reported. The present study examined whether EP ethylacetate extract(EPE) improves memory impairment induced by scopolamine. EPE (50 or 100 mg/kg) was administered orally to mice for 14 days, and then scopolamine (2 mg/kg, i.p.) was administered together with EPE for another 14 days. Memory performance was evaluated using the Morris water maze test and passive avoidance test. Also, brain acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activities, biomarkers of oxidative stress and the loss of neuronal cells in hippocampus was evaluated by histological examinations. Administration of EPE significantly restored memory impairments induced by scopolamine, as demonstrated by the Morris water maze test (escape latency and number of crossing platform area), and in the passive avoidance test. Treatment with EPE inhibited the AChE activity and increased the ChAT activity in the brain of memory impaired mice induced by scopolamine. Additionally, the administration of EPE significantly prevented the increase in lipid peroxidation and the decrease in GSH levels in brain of mice treated with scopolamine. Also, the EPE treatment restored the activities of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione reductase (GR) to control level. Furthermore, scopolamine‐induced oxidative damage of neurons in hippocampal CA1 and CA3 regions were prevented by EPE treatment. It is suggested that EPE may be a useful for mitigating memory improvement via the regulation of cholinergic marker enzyme activities and the suppression of oxidative damage of neurons in the brain of mice treated with scopolamine.
Epinephrine Increases Middle Cerebral Artery Blood Velocity in Response to Progressive Central Hypovolemia in Healthy Humans
Authors: Vo Christina C. , Joyner Michael J. , Curry Timothy B. , Convertino Victor A. , Baker Sarah E.
Published: April 2020

Objectives

We aimed to determine if epinephrine played a beneficial role on middle cerebral artery velocity (MCAv) during physiologically stressful stimuli prior to cardiovascular collapse. MCAv was assessed during lower body negative pressure (LBNP) under conditions of a saline or epinephrine infusion. We hypothesized that (1) epinephrine would augment the change in MCAv from baseline to LBNP tolerance, (2), epinephrine would increase MCAv for a given LBNP stimulus, (3) at a given percent of LBNP tolerance epinephrine and saline will be similar and (4) that epinephrine would improve LBNP tolerance.

Methods

Nine participants (35±9yrs, n=5 male, 4 female) underwent a LBNP protocol to physiological tolerance during two study visits, performed at least two weeks apart, and randomized to saline or epinephrine (dose=0.1μg/kg/min) infusions (peripherally‐ inserted central catheter). Blood pressure (radial artery catheter) and middle cerebral artery velocity (transcranial Doppler) were assessed. MCAv and cerebrovascular conductance index (CvCi) were analyzed during baseline (three minutes), physiological tolerance (one minute), during LBNP at a given stimulus (three minutes; −15, −30, −45 mmHg of LBNP), and during relative percentages of LBNP tolerance [three minutes; 20, 40, 60, and 80% of LBNP tolerance time (s)]. Baseline was defined as measures taken after 15 minutes of saline or epinephrine infusion.

Results

In both saline and epinephrine conditions, MCAv and CvCi decreased from baseline to tolerance (Saline MCAv: 69.7±7.5 vs 44.9±4.9 cm/s, p<0.05; Epinephrine MCAv: 80.9±5.9 vs 56.5±6.7 cm/s, p<0.05; Saline CvCi: 0.69±0.06 vs 0.58±0.067 cm/s/mmHg, p>0.05; Epinephrine CvCi: 0.80±0.06 vs 0.70±0.078 cm/s/mmHg, p<0.05). Epinephrine augmented MCAv at a given LBNP stimulus from baseline to −15, −30, and −45 mmHg (MCAv: Treatment effect: p<0.05; CvCi: Treatment effect: p=0.05). At a relative percent of LBNP tolerance, epinephrine increased MCAv, but when blood pressure was controlled for with CvCi there was no treatment effect between epinephrine and saline (MCAv: Treatment effect: p<0.05; CvCi: Treatment effect: p>0.05). Time to tolerance was not significantly different between saline and epinephrine conditions (1469±168 vs. 1445±82s, p=0.88).

Conclusions

Epinephrine increased MCAv during LBNP. These data suggest that epinephrine plays a beneficial role during stressful physiological stimuli such as hemorrhage in humans by augmenting cerebral blood flow prior to cardiovascular collapse. However, epinephrine did not improve LBNP tolerance.

Support or Funding Information

The United States Department of Defense W81XWH‐13‐2‐0038
An Innovative Approach For Demonstrating the Lymphatic System in the Gross Anatomy Laboratory
Authors: Lackey-Cornelison Wendy , Bauler Timothy
Published: April 2020

INTRODUCTION

From a gross anatomy perspective, the lymphatic system is difficult for students to observe and comprehend in the laboratory setting. Aside from the thoracic duct and spleen, many lymphatic structures are not often preserved well in embalmed cadavers or are damaged or destroyed during the dissection process. This presentation describes an intervention that was created to enhance gross anatomy learning of the lymphatic system.

AIM

To describe a case‐based anatomy laboratory approach that engages students and emphasizes the clinical relevance of gross anatomic knowledge of the lymphatic system.

INSTRUCTIONAL METHOD

Gross anatomy at Western Michigan University Homer Stryker MD SOM (WMed) is prosection‐based and taught within an integrated systems‐based curriculum. The laboratory session covering the lymphatic system occurs during the Immunology and Infectious Disease course. Organization of the session uses a combination of guided and cooperative learning techniques.

Student anatomy groups (6 students/group) progress through 7 cadaver stations, each having an associated clinical case. The cases encompass a variety of cancers, infections, and traumatic injuries to lymphatic system structures. At each station, students must identify the normal structures involved in the clinical scenario. In addition, students pin on the cadavers the location of lymph node groups that would likely be enlarged and/or need to be biopsied based on patient presentation and diagnoses. Cases also include short‐answer clinical application questions. Students within their groups work independently to identify and pin structures and compose their short answers. During the session, faculty circulate throughout the lab to confirm student answers and provide additional information when necessary.

RESULTS

Prior to this innovation, student feedback was below average and many students had difficulty seeing the relevance of the laboratory session. Since flipping the approach, evaluations have improved. Student comments expressing frustration over not being able to observe structures decreased. The number of comments expressing confusion about the purpose of the laboratory session and questioning if it is an effective use of time has also been reduced.

DISCUSSION AND CONCLUSION

This approach highlights the importance of the lymphatic system which is often overlooked in the gross anatomy laboratory. It provides faculty with a useful tool to help students see the clinical relevance of lymphatic gross anatomy and how this knowledge is applicable to physical examination and patient evaluation.

Support or Funding Information

There is no external funding for this project.
Identification and functional characterization of a biflavone as a novel inhibitor of TRPV4‐dependent proatherogenic processes in macrophages
Authors: Rahaman Shaik O. , Alharbi Mazen O. , Dutta Bidisha , Goswami Rishov
Published: April 2020

Atherosclerosis, a chronic inflammatory disease, is the major cause of mortality and morbidity in the United States. Studies suggest that arterial stiffness is a marker and a risk factor for atherosclerosis. Recent reports from our laboratory showed that TRPV4 (transient receptor potential channel of the vanilloid subfamily 4), a mechanosensitive ion channel, plays a role in oxidized LDL (oxLDL)‐induced macrophage foam cell formation, a critical process in atherogenesis. We performed a high‐throughput screening for antagonists of TRPV4 from a library of 2000 natural compounds using a fluorometric imaging plate reader (FLIPR)‐based Ca2+ influx assay. We identified ginkgetin, a biflavone, as a novel small molecule chemical inhibitor of TRPV4. Primary and secondary screening was performed with mouse bone marrow derived macrophages (BMDMs) to assess the ability of ginkgetin and other candidates to inhibit TRPV4‐dependent Ca2+ influx. In our hand’s, ginkgetin demonstrated an IC50 of 0.5 μM for inhibition of TRPV4‐dependent Ca2+ influx. Furthermore, we found that antagonism of TRPV4 by ginkgetin blocks oxLDL‐induced macrophage foam cell formation. Moreover, we found that ginkgetin treatment 1) attenuated oxLDL uptake but not its cell surface binding in BMDMs, 2) inhibited lipopolysaccharide‐induced phosphorylation of JNK, and 3) inhibited oxLDL‐induced expression of proinflammatory cytokines. Taken together, our results show that ginkgetin is a novel inhibitor of TRPV4‐mediated proatherogenic processes in macrophages.

Support or Funding Information

This work was supported by NIH (1R01EB024556‐01) and NSF (CMMI‐1662776) grants to Shaik O. Rahaman.
Identification of toxic metabolites produced by Batrachochytrium dendrobatidis fungus
Authors: Moore Lyndsey E.
Published: April 2020

Batrachochytrium dendrobatidis, or Bd, is a fungus that causes chytridiomycosis in amphibians, a disease responsible for the rapid decline in amphibian populations globally. The pervasiveness and mortality rates of the Bd fungus has spurred researchers to discover why the fungus is so lethal in various species of frogs, salamanders, and caecilians. While it is known that the fungus affects the skin of amphibians and causes death through suffocation via populating in the pores of adult amphibians, little is known about the active compounds in the fungus. An understanding of the methods of infection will aid in the creation of safe ways to treat amphibians and prevent further outbreaks. To determine the metabolites excreted by the fungus, the culture medium was filtered, dried, and analyzed by LCMS. A Daphnia assay was developed in order to aid bioactivity‐guided fractionation. Preliminary data suggests the presence of several compounds of interest in the dried metabolites. The masses of the peaks of interest were used to guide further isolation and identification of compounds.

Support or Funding Information

Funded by a SURF grant from the University of Tampa
A Microfluidics Approach to the High‐Throughput Generation of Quantifiable Cell‐Free Protein Synthesis Systems
Authors: Bashaw Benjamin T. , Peterson Andy T. , Schildknecht Kaitlyn T. , Tauchen Ali T. , Tittor Raechel T. , Gomez Paul , McKlin Sam , Chen Queena , Mazariegos Christopher , Zhou John , Seligmann Haley , Heyer Laurie L. , Campbell Malcolm , Poet Jeffrey L. , Eckdahl Todd T.
Published: April 2020

Synthetic biologists construct parts, devices and systems to engineer cells for applications in medicine, biofuels, chemical commodities, and the environment. A common problem with projects that focus on the synthesis of natural and engineered proteins is unpredictable translation directed by ribosomal binding sites (RBS). Researchers have hypothesized that base pairing interactions between RBSs and downstream coding sequences form secondary structures that affect the efficiency of translation initiation. Bicistronic designs (BCDs) were developed in 2013 by Drew Endy and his colleagues to enable more predictable and reliable in vivo translation. BCDs have an RBS that leads to the production of a leader polypeptide with no functionality and a second RBS that directs the production of a protein of interest. BCDs are better at producing predictable protein levels in bacterial cells than simple RBSs, thereby improving the ability of BCDs to function reliably and predictably as classic synthetic biology parts. In a companion study, we used GFP expression to compare the translational efficiencies of 19 BCDs during cell‐free protein synthesis (CFPS) to their efficiencies in vivo and found a rank correlation of 0.88. This report describes our efforts to expand the toolkit for CFPS protein production to microfluidic droplets. We used a microfluidic flow control system to rapidly produce nanoliter‐scale droplets and characterized them with microscopy. Image analysis of the droplet fluorescence intensities allowed us to quantify and compare BCD‐directed protein synthesis within droplets. We automated this process for many images to accelerate the workflow. We focused on three BCDs for CFPS of GFP in droplets and documented significant increases in observed fluorescence compared to batch CFPS outside of droplets. We also found that the rank order function for the three BCDs was the same in droplets as it was in batch CFPS. Our results support the use of BCDs to gain predictability of protein production in CFPS. These results set the stage for partitioning large libraries of gene regulatory combinations into microfluidic droplets to find the best combinations suited for a given synthetic biology application.

Support or Funding Information

NSF RUI MCB‐1613281 to Missouri Western State University, NSF RUI MCB‐1613203 to Davidson College
Venom peptides as selective inhibitors of bacterial ATP synthase
Authors: Ahmad Zulfiqar
Published: April 2020

Background

It is predicted that by the year 2050 world will face 10 million additional deaths per year due to antibiotic resistance. Misuse and overuse of antibiotics, is making microbes resistant to normally effective antibiotics. Therefore, it is of necessary to find different ways to combat microbial infections. ATP synthase is the main source of cellular energy production in almost all organisms from bacteria to men. Selective inhibition of bacterial ATP synthase to kill bacteria provides an effective way to eradicate antibiotic resistant bacteria. Venom peptides from many sources bind and inhibit ATP synthase. Venom peptides bind at the βDELSEED‐motif of ATP synthase. Although βDELSEED‐motif is highly conserved throughout the evolution but is skirted by more than twenty variable amino acids. We are identifying and characterizing the selective venom peptide inhibitors of ATP synthase using Escherichia coli as a model organism.

Method

Wild type, null, and mutant E. coli strains are used to select the strong venom peptide inhibitors of ATP synthase. Growth of E. coli strains is measured on fermentable and non‐fermentable carbon sources in presence and absence of venom peptides. F1Fo ATP synthase is isolated by harvesting cells in the minimal media, lysing cells by French Press, and isolating membrane bound ATP synthase by multiple high‐speed ultracentrifugation. A variety of venom peptides are used to inhibit ATP synthase. Inhibitory potency of venom peptides is augmented by incremental addition of charge and α helicity.

Results

Venom peptides cause variable degree of ATP synthase inhibition. Modifications of venom peptides cause increased inhibition. Addition of a c‐terminal NH2 group in venom peptides augment the inhibitory effects by about 100‐fold. Venom peptides also show synergistic inhibitory effects on ATP synthase.

Conclusions

Venom peptides potently kill E. coli cells by binding at βDELSEED‐motif of ATP synthase. ATP synthase can be used as a potent molecular drug target to combat antibiotic resistant bacterial infections.

Support or Funding Information

Warner/Fermaturo and ATSU Board of Trustees Research Grant
Immunolocalization of the Novel Protein FCHSD2 within Protrusive Actin‐Rich Bacterial Structures
Authors: Walker Brittany D. , Almeida-Souza Leonardo , Guttman Julian A.
Published: April 2020

Listeria monocytogenes and enteropathogenic E. coli (EPEC) both exploit clathrin mediated endocytosis and the actin cytoskeleton for their disease processes. During L. monocytogenes infections clathrin is used for the microbes to invade their host’s cells and actin filaments are used to generate actin‐rich comet/rocket tails that propel the bacteria within the host cell cytoplasm. When the bacteria hit the host plasma membrane, the comet/rocket tails push the bacteria into neighbouring cells by forming a membrane distention housing the bacteria and the actin tail which forms a corresponding invagination in the neighbouring cell for eventual cell‐to‐cell spreading of the bacteria. EPEC on the other hand remains extracellular and sit atop infected cells on actin‐rich pedestal‐like structures that also recruit clathrin to their apical regions. The FCH and double SH3 domains protein 2 (FCHSD2) functions to activate actin polymerization during clathrin mediated endocytosis. As both L. monocytogenes an EPEC utilize CME and actin polymerization we examined the localization of FCHSD2 during the infections. We found that during L. monocytogenes infections FCHSD2 was absent from the bacterial comet tails, but was prominent at L. monocytogenes actin‐rich membrane protrusions. During EPEC infections FCHSD2 was present within the actin core of the pedestals but did not colocalize with clathrin. Together, this work suggests the FCHSD2 protein has a distinct role at actin‐rich bacterial structures that interact at the membrane which is independent of clathrin.
Stress Promotes a More Invasive Phenotype in Human Melanoma Cells
Authors: Amomoy Jerry , Barkoudeh Laila , Phung Vanessa , Nguyen Cecilia , Fajardo Abigail , Zarghooni Melody , Rosero Rebecca , Klettke Elizabeth , Villares Gabriel J.
Published: April 2020

Growing evidence suggests that stress plays a vital role in metastasis and tumor development by activating the sympathetic nervous system. The catecholamines released into the tumor microenvironment (TME), specifically norepinephrine (NE), results in a cascade effect leading to a variety of pro‐metastatic activities that sustain tumor growth and increase melanoma aggressiveness. Most notable are the secretion of cytokines and the stimulation of tumor‐associated macrophages (TAMs). Using a co‐culture system of melanoma cancer cells and macrophages we found that sustained exposure of NE in the microenvironment induced the release of tumor‐growth and progression‐associated cytokines from TAMs. Of the upregulated cytokines found; IL‐11, IL‐24, GRO‐a, DKK‐1, and angiopoietin‐2 are known to induce growth, migration, invasion, and tumor development in human melanoma. Our next steps include validating the cytokine array using real time PCR and using an ELISA assay to quantify protein levels of antigens associated with the cytokines secreted from the cells and macrophages.

Support or Funding Information

US Department of Education for the HSI‐STEM Grant University of St. Thomas for Student Research for partial funding
Using Interactive Technology in an Integrated Human Physiology and Functional Anatomy Course to Enhance Student Engagement, Knowledge, and Retention
Authors: Stokes Jennifer Ann , Silverthorn Dee
Published: April 2020

The curriculum for a traditional two‐semester anatomy and physiology (A&P) course was revised into an integrated two‐semester physiology and functional anatomy course for undergraduate students going into the health professions. The revised curriculum implemented the use of digital learning technology with the cloud‐based ADInstruments (ADI) Lt learning platform. This platform provides students in the classroom and laboratory with interactive and immersive experiences aimed at enhancing knowledge retention and practical application. Students enrolled in the first semester (fall 2019) of the two‐semester course sequence completed individual lessons and group lab activities throughout the semester using ADI‐supplied Lt lessons with instructor modifications. The goals of the individual lessons were to provide students with opportunities to review the difficult course concepts, allowing additional exposure to the content in a new format, and opportunities to practice applying the content to clinical scenarios. Students received immediate feedback on their answers as part of the lesson. The goals of the group lab activities were to provide the students with the opportunity to apply course knowledge to a real‐life physiological scenario, formulate a hypothesis, record and analyze data, and draw conclusions. Group lab activities included analysis of heart rate, muscle contraction and EMG, grip strength and fatigue, and a guided heart dissection. Student surveys assessed student perceptions and engagement with the ADI Lt system. Summative assessments of student knowledge and retention included unit exams, a cumulative final exam, and lab practical exams. Summative assessment data were compared to previous course cohorts. Data collection followed guidelines approved by the Centenary College Institutional Review Board (Protocol #18‐002).

Support or Funding Information

This project is supported by the Teaching Career Enhancement Award from The American Physiological Society.
ΔNp63α suppresses cell invasion through downregulating Rac1 activity
Authors: Aljagthmi Amjad Ahmed , Cooke Mariana , Kazanietz Marcelo , Kadakia Madhavi P.
Published: April 2020

ΔNp63α, a member of the p53 family of transcription factors, is overexpressed in a number of cancers and plays a role in proliferation, differentiation, migration and invasion. We showed previously that ΔNp63α suppresses cell invasion by positively regulating miR‐320a, resulting in the downregulation of its direct target protein kinase C gamma (PKCg) and a corresponding reduction in phosphorylated Ras‐related C3 botulinum toxin substrate 1 (Rac1). Here, we further show that ΔNp63α inhibits Rac1 GTP activity. Using a Rac1 pulldown assay to measure Rac1‐GTP levels, we showed that silencing ΔNp63α in A431 cells led to a significant upregulation of Rac1‐GTP. Silencing ΔNp63α in A431 cells also led to an increase in p21‐activated kinase (PAK1) phosphorylation, confirming the negative regulation of ΔNp63α on Rac1. Inhibiting Rac1 GTP activity using Ehop‐016 led to a significant reduction in both pRac1 and pPAK1 levels. Treatment with Ehop‐016 reversed the increase in cell invasion observed upon silencing ΔNp63α. RT‐PCR analysis performed on an array of 43 Rac guanine exchange factors (Rac‐GEFs) showed that 6 GEFs are significantly upregulated by silencing ΔNp63α in A431 cells. Taken together, our data suggest that ΔNp63α negatively regulates Rac1 GTP activity via downregulation of a Rac‐GEF, thereby reducing Rac1‐GTP levels and inhibiting cancer cell invasion.

Support or Funding Information

This work was supported by a grant from the National Cancer Institute [1R01CA154715] to M.P.K.
Production of Ethanol from Lactose in Engineered S. cerevisiae
Authors: Vinz Cami Alyssa
Published: April 2020

Wisconsin is the dairy capitol of the United States. However, all this cheese production leaves behind a lot of unused by product in the form of cheese whey. If cheese whey could be fermented into consumable ethanol there would be less discarded into the trash and could also be a new revenue stream for Wisconsin dairies. Our plan is to modify yeast that can already produce ethanol from sucrose to also be able to make the ethanol from cheese whey that contains lactose.

By engineering two strains of Saccharomyces cerevisiae to metabolize lactose we predict they will be able to digest and ferment cheese whey. This is done by allowing the yeast to express lactose permease and beta galactosidase, LAC12 and LAC4 respectively. These genes can be found in Kluyveromyces marxianus and Kluyveromyces lactis.

With this production of ethanol from cheese whey we plan to patent this newly found technique which in return will lead to lots of vodka and money.

Support or Funding Information

This project is funded by UW ‐ Stout's Office of Research and Sponsored Programs.
Evaluation of Virus‐based Vectors for Cystic Fibrosis Gene Therapy
Authors: Rodríguez-Villafañe Andrea , Kuntz Leah , Limberis Maria P.
Published: April 2020

Cystic fibrosis (CF) is a life‐threatening genetic disease caused by mutations in the CF transmembrane conductance regulator gene. It is the most common genetic disease among Caucasians. There is an urgent need to create more therapies for CF since current drugs are not effective in treating all the mutations that cause this disease. Furthermore, gene therapy is a potential treatment for CF airway disease. Because of this, various viral‐based gene transfer vectors have been evaluated for their efficiency in correcting the CF airway disease phenotype. This project aimed to compare side‐by‐side the differences in the transduction efficiency and targeting of specific cell types of adenovirus (Ad)‐based and adeno‐associated virus (AAV)‐based vectors in different models of the airway. Different serotypes of AAV, namely 1, 2, 5, 6, and Ad5‐based vectors, expressing green fluorescent protein (GFP), firefly luciferase (ffLuc) or ß‐galactosidase (LacZ) were assessed in in vitro and in vivo gene transfer studies. Madin‐Darby Canine Kidney (MDCK) cells were grown submerged on plastic, and human airway epithelial (HAE) cells were grown on transwells and fed only from the basolateral side to attempt to model the human conducting airways. The viral vectors expressing GFP were tested at doses of 102–103 genome copies (GC) for AAV or plaque‐forming unit (pfu) for Ad, and the level of transduction was assessed at different time points. The data demonstrates that in MDCK and HAE cells, the highest transduction was achieved by the Ad5 vector followed by the AAV2 vector, while AAV6 had the lowest transduction. Additionally, as examined in HAE cells, the age of the culture impacts transduction efficiency. In parallel, AAV vectors 1, 2, 5, 6 (at a dose of 8×1010 – 3×1011 GC) and Ad5 vector (at a dose of 1×1011 pfu) expressing ffLuc or LacZ were intranasally delivered to mice to assess the transduction efficiency in the epithelium of both the nose and lungs. AAV6 was the most efficient at transducing the cells of the nasal epithelium of mice and AAV5, at transducing lung cells. AAV2 did not transduce airway cells in mice. In conclusion, we confirmed that the viral vectors have different transduction profiles in different models of airway epithelium. More airway models should be studied to better understand which are more useful for preclinical assessments of CF gene therapeutics.

Support or Funding Information

This project was supported by the National Heart, Lung, and Blood Institute (R25‐HL084665‐14).