bluuebunny/crossref_metadata_2025 huggingface dataset:
Adaptive Estimation for Epidemic Renewal and Phylogenetic Skyline Models
Authors: Parag Kris V , Donnelly Christl A
Published: April 2020

Estimating temporal changes in a target population from phylogenetic or count data is an important problem in ecology and epidemiology. Reliable estimates can provide key insights into the climatic and biological drivers influencing the diversity or structure of that population and evidence hypotheses concerning its future growth or decline. In infectious disease applications, the individuals infected across an epidemic form the target population. The renewal model estimates the effective reproduction number, R, of the epidemic from counts of observed incident cases. The skyline model infers the effective population size, N, underlying a phylogeny of sequences sampled from that epidemic. Practically, R measures ongoing epidemic growth while N informs on historical caseload. While both models solve distinct problems, the reliability of their estimates depends on p-dimensional piecewise-constant functions. If p is misspecified, the model might underfit significant changes or overfit noise and promote a spurious understanding of the epidemic, which might misguide intervention policies or misinform forecasts. Surprisingly, no transparent yet principled approach for optimizing p exists. Usually, p is heuristically set, or obscurely controlled via complex algorithms. We present a computable and interpretable p-selection method based on the minimum description length (MDL) formalism of information theory. Unlike many standard model selection techniques, MDL accounts for the additional statistical complexity induced by how parameters interact. As a result, our method optimizes p so that R and N estimates properly and meaningfully adapt to available data. It also outperforms comparable Akaike and Bayesian information criteria on several classification problems, given minimal knowledge of the parameter space, and exposes statistical similarities among renewal, skyline, and other models in biology. Rigorous and interpretable model selection is necessary if trustworthy and justifiable conclusions are to be drawn from piecewise models. [Coalescent processes; epidemiology; information theory; model selection; phylodynamics; renewal models; skyline plots]
Structural and Interaction Insight in Streptococcal beta C Proteins
Authors: Xu Xin , van Sorge Nina , van der Lans Sjors , van Woudenbergh Esther , van Strijp Jos , McCarthy Alex J , Geisbrecht Brian
Published: April 2020

Group B Streptococcus (GBS) is a leading cause of invasive bacterial infections in human newborns. The study of GBS protein is important for understanding its pathogenesis and epidemiology of infection. To date, several GBS surface proteins with the ability to induce protective antibodies in animals have been reported, including beta C proteins. The beta C protein is a GBS surface protein being investigated as a potential vaccine candidate. Here, we constructed truncated beta C proteins in order to study the function of this protein. Factor H (fH) is involved in the process of complement activation and is a complement control protein. Its principal function is to regulate the alternative pathway of the complement system, playing a role in ensuring the complement system is directed towards pathogens rather than host cells. While it was previously reported that GBS binds to fH, how this occurs at the molecular level has remained unclear. We used truncated beta C proteins to map the interaction between fH and beta C to a defined region within the beta C molecule. In addition, we have determined a 2.36 Å resolution crystal structure of this portion of beta C, which reveals a variation upon the alpha helical bundle fold common in Gram‐positive innate immune evasion proteins. These findings enhance our knowledge of a major cell wall‐anchored surface protein of GBS and promote our understanding of its role in the pathogenesis of streptococcal disease.
The Bradykinin B2 Receptor Agonist NG291 Causes Rapid, Transient Blood‐Brain Barrier Disruption Without Evidence of Persistent Brain Injury: Implications for Drug Delivery to the Brain.
Authors: Masso Sergio Rafael Rodriguez , Erickson Michelle , Banks William A. , Martins Antonio Henrique
Published: April 2020

The blood brain barrier (BBB) is a term used to describe the highly specialized capillaries of the brain which forms a dynamic interface that maintains central nervous system (CNS) homeostasis. The BBB supports the CNS, in part, through its barrier functions which prevent entry of potentially harmful circulating molecules into the brain. However, the barrier activities of the BBB are also an obstacle to the delivery of therapeutics to the brain. One strategy to facilitate the delivery of therapeutics into the brain is to transiently induce BBB disruption. Bradykinins are one class of molecule produced endogenously and contribute to transient BBB disruption through agonist activity on the bradykinin B2 receptor (B2R).Unfortunately, the endogenous peptide bradykinin is of little therapeutic value because of its short half‐life and because it also activates the bradykinin B1 receptor that generates an exaggerated inflammatory response. We have been studying NG291, a physiologically stable bradykinin analogue that has selective agonist activity on B2R, and its ability to disrupt the BBB. Sprague Dawley (SD) rats and CD‐1 mice were subjected to NG291 treatment (either 50μg/Kg or 100μg/Kg, IV). BBB disruption was measured by Evans blue extravasation in SD rats in a time and dose dependent fashion. Disruption was evaluated in mice using the large and small sized radioactive tracers 99mTc‐Albumin (66.5KDa) and 14C‐Sucrose (340Da), respectively. We evaluated the potential harm of NG291 administration in rats via changes in brain water content and detection of cellular death via Fluoro‐Jade C staining. Evans blue studies in rats confirmed that NG291 is disrupting the BBB in a time and dose dependent fashion with the BBB regaining function 2hr after 50μg/Kg and 4hr after 100μg/Kg of NG291 administration. In mice, both albumin and sucrose tissue/serum ratio were significantly increased within 15 minutes of NG291 injection vs vehicle controls. We have observed no increased cell death nor change in water content in groups treated with NG291 when compared to saline injected controls. We conclude that NG291 has the potential to acutely disrupt the BBB, generating a window where potential CNS therapies can be more effectively delivered to the brain. This has the potential to speed up drug development process, increase the number of CNS therapies that reach the market, and help those in dire need of such treatments.
Tunicamycin Interferes With Wnt Signaling and Inhibits Angiogenesis
Authors: Cruz Melanie
Published: April 2020

Wnt signaling supports cellular proliferation. The transcription co‐author β‐catenin is also associated with the tissue development. Recent studies have shown Wnt signaling plays a significant role in vascular development, an important event in tumor progression. Our laboratory has established protein N‐glycosylation inhibitor, Tunicamycin reduces angiogenesis. It also reduces the progression of ER−/PR−/HER2+ and ER−/PR−/HER2− breast tumors in athymic nude mice. Tunicamycin is a competitive inhibitor of N‐acetylglucosaminyl 1‐phosphate transferase (GPT) activity and down regulates Glc3Man9GlcNAc2‐PP‐Dol biosynthesis. The consequence is “ER stress” and the cell cycle arrest in G1. We have then hypothesized that Tunicamycin negatively impacts Wnt signaling and the objective is to delineate this dynamic relationship. We have used western blotting, immunofluorescence microscopy and capillary endothelial cells as a model, and observed down regulated expression of Wnt proteins. We, therefore, conclude that Tunicamycin‐induced ER interferes with the Wnt signaling and inhibits angiogenesis.
The Effect of the Short‐Term Growth Hormone Administration on Non‐Alcoholic Fatty Liver Disease in Diet‐Induced Obesity Models
Authors: Likitnukul Sutharinee , Thammacharoen Sumpun , Kalandakanond-Thongsong Sarinee , Sriwatananukulkit Orada , Hemstapat Ruedee , Pinthong Darawan
Published: April 2020

Non‐alcoholic fatty liver disease (NAFLD) is one of the characteristics of metabolic syndrome. Most people are facing NAFLD whether they are diagnosed with obesity or not. Additionally, growth hormone (GH) administration could mediate the storage of body fat, including in the liver, as a result of the lipolytic effect. In obese patients, the reduction of GH level has been reported. The long‐term treatment of obesity with GH has been shown to reduce body fat mass as well as its well‐known effect to counteract insulin action, leading to insulin resistance. However, this remains unknown whether the short‐term GH administration can alter the fat storage in the liver while it does not worsen insulin sensitivity. To investigate this hypothesis, the rats were fed with either high‐fat (HF) or standard diet for 6 weeks, resulting in diet‐induced obesity (DIO) or diet‐resistant (DR) rats. The effect of short‐term GH treatment (1 mg/kg, twice daily, subcutaneous injection) was then assessed and compared to saline treatment after 3‐day of GH administration. A glucose tolerance test was performed after GH treatment to assess insulin sensitivity in vivo. After euthanasia, the liver was weighed and collected to measure triglyceride concentration by colorimetric assay. Blood was also collected to determine plasma free fatty acid (FFA) levels for the measurement of the lipolytic activity of GH. The results revealed that the percentage of liver mass was significantly greater in DIO and DR rats compared to control rats (p<0.05). The short‐term GH administration did not alter the percentage of liver weight. For liver triglyceride level, DIO and DR rats had a higher level than that of control rats (p<0.05); however, the liver triglyceride level did not affect by the short‐term GH treatment in all rats. Interestingly, the short‐term GH administration significantly increased plasma FFA only in DR rats (p<0.05). Moreover, the effect of GH significantly improved glucose tolerance especially in DIO rats (p<0.01), whereas it did not significantly affect insulin sensitivity in control and DR rats. Taken together, this study suggested that the short‐term GH administration may influence liver lipid metabolism in DR rats. However, further studies are required to determine whether short‐term GH administration affected liver lipid metabolism by interfering with the gene expression, including lipid uptake, lipid synthesis, and lipid oxidation. Additionally, the mechanisms underlying the effect of the short‐term GH administration on insulin sensitivity remains the subject for further studies.
BDNF‐Induced Arc Expression Was Enhanced by PIC in Neuronal Cells Treated with High Glucose and Amyloid Beta
Authors: CHA HEEYUN , Kim Se Lin , Heo Sumin , Park Sol Been , Han Yeo Gyeong , Yang Soo Jin
Published: April 2020

Objectives

Hyperglycemia has been shown to be associated with Alzheimer’s disease and impairment of cognitive function. Alzheimer’s disease is characterized by the excessive formation of amyloid‐beta (Aβ) and neurofibrillary tangle. Aβ is generated by cutting amyloid precursor protein (APP) by β‐secretase (BACE1) and γ‐secretase. Brain‐derived neurotrophic factor (BDNF) is the important molecule that is involved in synaptic plasticity and neuronal survival. BDNF binds to trkB receptor and activates PI3K/Akt/mTOR pathway, and it increases synaptic efficacy by inducing expression of activity‐regulated cytoskeleton‐associated protein (Arc) which is the marker of plastic changes of the brain. Suppression of BDNF is related to the pathogenesis of Alzheimer’s disease. Piceatannol (PIC) is a polyphenolic compound that has similar structure to resveratrol having neuroprotective efficacy. Thus, we investigated the effects of PIC treatment on amyloidogenesis and BDNF signaling in neuronal cells treated with high glucose and Aβ.

Methods

The SH‐SY5Y cells were treated with 25 mM D‐glucose and 10 μM Aβ in order to induce the cell culture model of high glucose‐induced Alzheimer’s disease. And then, cells were treated with 20 μM PIC for 24 h. Relative gene expression levels of amyloidogenesis‐ and BDNF signaling‐related markers were analyzed. Aβ concentrations were measured by ELISA to examine whether PIC alters the levels of Aβ deposit. One‐way analysis of variance (ANOVA) was performed to assign statistical significance followed by Fisher’s Least Significant Difference (LSD) post‐hoc analysis using SPSS software (IBM SPSS Statistics 24).

Results

In the condition of high glucose‐induced Alzheimer’s disease, PIC treatment significantly increased the relative gene expression of BACE1 (P = 0.003). The presenilin 1 expression tended to rise up by PIC in this condition. The concentrations of Aβ were significantly lower in cells treated with high glucose, Aβ, and PIC compared with those without PIC (P < 0.001). The relative expression levels of BDNF in high glucose group treated with PIC tended to be higher than those without PIC. PIC treatment significantly increased the relative gene expression of BDNF in cells treated with high glucose and Aβ (P = 0.002). The Arc expression was also significantly increased by PIC in those cells (P = 0.038).

Conclusions

We found that PIC improves the pathological alterations by high glucose and Aβ, which is accompanied by decreased Aβ formation and upregulation of BDNF and Arc expressions in high glucose and Aβ‐treated neuronal cells. Therefore, PIC may have a therapeutic effect on the pathological conditions of hyperglycemia‐induced Alzheimer’s disease.
Student Attention in the Modern Classroom: An Eye‐Tracking Field Study
Authors: Santiago Kevin , Wilson Timothy D.
Published: April 2020

Background

Multitasking, commonly perceived to be a method of increasing productivity and maximizing time, is increasingly commonplace among student populations. However, considering recent work examining split attention and the cognitive demands of multitasking behaviour, multitasking is negatively associated with several key learning outcomes in laboratory environments such as information retention, comprehension and academic performance. To better characterize multitasking behaviours in modern classrooms, a field study was conducted using self‐reported student data coupled with gaze behaviour on attention during lectures. Specifically, this study contrasts graduate (GS) and undergraduate (UGS) students’ self‐assessments to in‐class attention to explore whether students are self‐aware of multitasking behaviours.

Methods

An online questionnaire was developed and distributed to a large mixed didactic anatomy class of GS and UGS. The survey was an amalgamation of three validated questionnaires, Media Use Questionnaire, Media Multitasking Class Index (MMCI), and the Attitudes Scale towards multitasking behaviours. In addition, a subset of students (GS & UGS) wore a single‐eye gaze tracking device during 2 lectures (Arrington Research Viewpoint) throughout the fall term. Students’ (n=4) visual fields were characterized as an index of attention to On‐task (course‐related) and Off‐task (non‐course related) activities. The gaze‐tracked location and duration of multitasking activities were identified off‐line and normalized to a percentage of class time to characterize the extent to which students engage in On‐ and Off‐task activities during lecture.

Results

Initial analysis of the MMCI survey section (n=105) indicates GS (n=69) estimated spending 7% less class time on on‐task activities than UGS (n=36) (74±0.2 vs 81±0.1, p < 0.02). Gaze tracking indicates GS (n=1) spent 37% more time engaging in multitasking (off‐task) behaviours than UGS (n=3) (45% vs 8% respectively). The UGS MMCI off‐task behaviours compared to that cohort’s gaze tracking suggests students estimate their multitasking behaviours accurately in the classroom (7±0.1% vs 8±0.0%). Through further analysis of attentional time dedicated to on‐task activities (75±0.0%), students largely focused on their notes (54±0.1%), multimedia presentation (11±0.1%), and then the professor (0.4±0.0%) of total class time. Of the time spent in off‐task activities (8±0.0%), distractors to attention included activities such as doodling, adjusting clothes and observing neighbours (7±0.0%), text messaging (0.3±0.0%), and consuming food and/or liquids (0.3±0.0%).

This is the first field study of this kind indicating how multitasking is a part of student behaviour and how attention is allocated in the modern anatomy lecture hall through gaze‐tracking. As midterm grades become available, we will correlate both real and perceived behaviours to class performance.
Peripherally Restricted Dual Cannabinoid Receptor Agonist Modifies Mitochondrial Signaling and Inhibits Atrial Electrical Remodeling in a Tachypaced Ex Vivo Rat Model
Authors: Lee Danielle I. , Murninkas Michael , Elyagon Sigal , Etzion Yoram , Anderson Hope D.
Published: April 2020

Atrial fibrillation (AF) leads to rate‐dependent changes, which progressively increase the risk for recurrence and persistence of the arrhythmia (collectively defined as atrial remodelling). Atrial effective refractory period (AERP) shortening and conduction velocity prolongation are hallmarks of atrial remodelling markedly promoting AF substrate. The molecular mechanisms underlying atrial remodelling are not fully elucidated and therapeutic strategies to reduce AF burden by preventing atrial remodelling are not yet available. Cannabinoid (CB) receptor ligands exert cardioprotective effects. We hypothesized that CB13, a peripherally restricted dual CB receptor agonist with limited brain penetration, would reduce atrial remodelling.

Sprague Dawley (SD) rat hearts were excised and hung on a Langendorff set‐up, and supplemented with CB13 (1μM) or without (control) (paced and non‐paced). Coronary pressure of 60–80 mmHg was maintained. Electrophysiological (EP) parameters were measured by placing a mini‐hook platinum‐iridium quadrupole electrode on the right atrium (RA) for pacing and atrial recording (CB13 n=8; control n=8). RA were tachypaced for 90 min at double diastolic threshold and a cycle length of 5 ms higher than failed 1:1 atrial capture. S1S2 stimulation protocol was used to measure AERP, defined as the longest interval with failed 1:1 capture. The effects of CB13 on atrial refractoriness and AF substrate were assessed by analysis of high resolution electrogram recordings and protein expression via western blot.

Post‐tachypacing EP recordings showed reduced AERP in control compared to pre‐tachypacing (54.38±3.05 vs. 44.75±3.67 ms; p<0.01). AERP post‐tachypacing in CB13‐treated hearts remained unchanged compared to pre‐tachypacing (55.25±3.46 vs. 55.50±2.67 ms; ns). Thus, CB13‐treatment resulted in preservation of AERP compared to control (103.6±9.2 vs. 82.3±4.4 % of baseline; p<0.05). Protein expression of PGC1α was significantly increased by CB13 compared to controls (paced and non‐paced). Upregulation of mitochondrial biogenesis may be related to increased phosphorylation of AMPKα at residue threonine 172 (compared to total AMPKα, p=0.12, CB13 vs. control paced). Furthermore, gap junction connexin 43 was significantly downregulated in paced controls vs. non‐paced controls, while CB13 remained upregulated (no significance vs. controls).

CB13 exposure during tachypacing prevents AERP reduction and gap junction downregulation probably through mechanisms involving mitochondrial biogenesis. Thus, by inhibiting atrial remodeling CB13 and the subsequent activation of CB receptors may be a viable treatment strategy for AF.

Support or Funding Information

This work was supported by studentships from the Canadian Institutes of Health Research and Mitacs (D.L.)
Characterization of Collagen Metabolism in Normal and Chronic Obstructive Pulmonary Diseased Human Lung Fibroblasts Reared in Serum‐free Medium
Authors: Otto Ashley Maria , Mattioli Patrisia , Musiol Joseph , Popper Hannah , Graham Morgan , Laird Patrick , Maloney Trevor J. , Geary Kyla , Daghigh Farzaneh , Borghaei Ruth , D’Angelo Marina
Published: April 2020

The structural integrity of the human lung is maintained by a healthy extracellular matrix (ECM). One of the main components, collagen type I is produced by fibroblasts and provides strength and structure to the cells and the function of the lung. A careful balance of collagen degradation and production is controlled by matrix metalloproteases (MMPs). In diseased states such as Chronic Obstructive Pulmonary Disease (COPD), two collagenases, MMP‐8 and MMP‐12, are upregulated. In this study we measure collagen metabolism of human lung fibroblasts (HLFs) reared in serum‐free media (subconfluent normal (NHLF) and COPD diseased (DHLF)). Commercially available early passage human lung fibroblasts established from lung tissue, obtained during biopsy, were cultured and passaged in our laboratory through P4 and P5 for both the NHLF and DHLF. Both normal and diseased HLF cells were viable when cultured at subconfluence for 24 hours in serum free medium. They produced detectable quantities of intact collagen type I secreted and incorporated in the ECM. The amount of intact collagen decreased between passage 4 and 5 (p<0.01) in both cell types indicating there is a limit to the passages that can be utilized to measure ECM alterations. Degraded collagen type I was present in the cell layer fractions of both cell types, but was 10 fold higher in the cell layer of diseased HLF cells. This indicates that the expected ECM profile of diseased HLF can be demonstrated in serum‐free culture. Both cell types produced detectable levels of MMP‐8 and MMP‐12 collagenases. In this study we have demonstrated that normal and diseased HLF can be cultured in serum‐free conditions while maintaining their metabolic profile. The production of MMPs and collagens provides an experimental system in which we can test potential therapeutics that may alter the fibrotic response during COPD.

Support or Funding Information

This work is supported by a grant from the PCOM Alumni and the Division of Research at PCOM.
Impaired Female Reproductive Function in A Novel Estrogen Receptor Alpha Hypomorphic Mouse Model
Authors: Saito Kenji , Rodeghiero Samuel R. , Dickey Jacob E. , Paradee William J. , Cui Huxing
Published: April 2020

Introduction

Estrogen signaling plays an important role in the regulation of both energy homeostasis and reproductive function. Human and animal studies indicate that the metabolic actions of estrogen signaling are primarily mediated by estrogen receptor alpha (ERα). Both human and mouse ERα genes show remarkable heterogeneity due to complicated alternative splicing, which results in a wide variety of variant transcripts with different protein products or a common protein. However, functional significance of this ERα genomic complexity has not been well documented. Here we generated a novel mouse model in which ERα expression was differentially affected in various tissues, and examined their body weight change and reproduction.

Method

Using CRISPR/Cas9 technology, we introduced a loxP‐flanked sequence containing En2 splice acceptor, a green fluorescent marker with mitochondrial localization signal and DNA translation stop signals before the first coding exon of mouse ERα gene for the potential restoration of the ERα expression.

Results

ERα protein expressions were significantly reduced in pituitary (~37%) and ovary (~67%), but not in brain, of the homozygous mutant female compared to those of controls, indicating that unknown elements important for ERα tissue‐specific expression were affected. In contrast to body weight gain observed in previous studies of ERα knockout female mice, our hypomorphic mutant females did not show significant body weight gain. On the other hand, virginal smears from the mutant females rarely showed typical estrus stage. Uterine wet weight was significantly lower in the mutant female mice compared to the controls, indicating abnormal reproductive system. Continuous mating study further confirmed delayed pregnancy and inability to have alive newborns in the mutant female mice.

Conclusion

Estrogen receptor alpha protein expression varies among tissues in terms of the level and pattern of splicing variants. Splicing variant‐specific ERα functions are not fully elucidated. Our results suggest that a particular variant affected in our mouse model is less critical for proper functioning of systems involved in estrogen‐regulated energy metabolism, but indispensable for some aspects of female reproductive function.
Inhibition of Oxidative DNA Damage in the Presence of Spinach Extract
Authors: Lezo Daisy , Ramirez Ashley , Stemp Eric
Published: April 2020

Oxidative damage can induce DNA protein cross‐linking. The present study aimed to evaluate whether aqueous extracts of spinach can inhibit oxidative cross‐linking between DNA and protein. In the flash‐quench method, the intercalator Ru(phen)2dppz2+ (phen = phenanthroline, dppz = dipyridophenazine) is excited with blue light, releasing an electron to the quencher, Co(NH3)5Cl2+. The oxidized intercalator then extracts an electron from guanine and the guanine radical reacts to produce DNA‐protein cross‐links. Samples containing Co(NH3)5Cl2+, histone, Ru(phen)2dppz2+, and DNA were subjected to flash quench treatment in the absence or presence of a spinach extract. Cross‐linking was then detected in two ways—chloroform extraction assay and gel shift assays. With calf thymus DNA, the chloroform extraction assay was used, and this UV spectroscopy method showed that the spinach extract significantly decreased the loss of the free DNA absorbance due to crosslinking visible in a spectra and a plot of % crosslinked vs time. In gel shift assays, pUC19 DNA subjected to the flash quench technique decreased greatly in mobility, with a considerable amount of cross‐linked material visible within the wells. In the presence of an aqueous extract of spinach, cross‐linking is strongly inhibited and the DNA runs as a single band. This inhibition is maintained out to significant dilutions of the green extract. More recently, we have examined kaempferol, one antioxidant found in spinach, and have observed protection of DNA via a nicking assay. A single component of spinach would not be a universal protector against oxidative stress‐induced disease. Lipophilic components are also abundant, which is why two of them, vitamin K and β‐Carotene, are currently being examined.
SPARC: Effect of Waveform on Kilohertz Frequency Nerve Block
Authors: Pena Edgar , Pelot Nicole A. , Grill Warren M.
Published: April 2020

There is growing interest in delivering kilohertz frequency (KHF) electrical signals to block conduction in autonomic nerves as a therapy to treat various diseases. Previous computational modeling, preclinical, and clinical studies used multiple different KHF waveforms to achieve block of the sciatic, vagus, and pudendal nerves. However, it remains unclear how the choice of waveform affects clinically relevant nerve block parameters. To address this knowledge gap, we quantified the effects of waveform on KHF block in vivo for the rat sciatic nerve, as well as in computational models of the rat sciatic nerve and the human vagus nerve. We compared block thresholds and onset responses across KHF sinusoids and charge‐balanced rectangular waveforms with different symmetries and duty cycles.

Block thresholds were strongly dependent on the waveform shape. Experimentally, symmetric rectangular waveforms with full duty cycles had lower current thresholds for block than sinusoids, but they used slightly more power at threshold. Further, lower duty cycles yielded higher block thresholds, such that the log‐transformed block thresholds increased in an approximately linear manner as duty cycle decreased. The results from computational models were consistent with the in vivo block threshold relationships, although the models underestimated the increase in threshold with decreased duty cycle.

While waveform selection had a clear and distinct effect on thresholds, it did not consistently affect in vivo onset response. Onset magnitude was similar across waveforms when normalized to block threshold. We also observed a history‐dependent reduction in onset response across all waveforms, from earlier trials with larger onset responses to later trials with reduced onsets during the course of a given experiment.

Waveform had substantial effects on block thresholds but not on onset response. These quantitative data inform choice of waveform in subsequent studies and clinical applications. Such an understanding of the direct effects of KHF signal parameters on neural activity will enhance its effective use in therapeutic applications and facilitate the design of KHF stimulation parameters that achieve block with minimal undesired onset responses.

Support or Funding Information

NIH SPARC OT2 OD025340
In‐Vivo Knockdown of Cortactin Disrupts Basal Tubulobulbar Complexes and Results in the Presence of Redundant Junction Related Ectoplasmic Specializations
Authors: Adams Arlo , Palia Prunveer , Vogl Wayne
Published: April 2020

In the mammalian testis, spermatogenic cells begin development in the germ cell niche or basal compartment of the seminiferous epithelium. They then translocate through massive junction complexes between neighboring Sertoli cells to enter the adluminal compartment and further differentiate. Removal of the junctions above and formation of new junctions below is necessary for the translocation of cells from one compartment to the other. Tubulobulbar complexes’ (TBCs) are Sertoli cell specific structures that are proposed to internalize intercellular junctions in the seminiferous epithelium. TBCs are actin cuffed endocytic structures that share many features in common with clathrin mediated endocytosis (CME). Both TBCs and CME vesicles begin with the association of a clathrin coated pit on the plasma membrane and the development of dendritic actin ‘cuffs’ on their neck regions. Rather than vesiculating, the necks of TBCs continue to lengthen into microns long structures, eventually developing an actin free ‘bulb’ region, which develops an ORP9 positive membrane contact site with an associated leaflet of endoplasmic reticulum. One of the proteins known to be highly specific for the dendritic actin around TBCs is the actin related protein, cortactin. It previously has been shown that knockdown of cortactin causes shortening and disorganization of those TBCs at the apex of the epithelium that are associated with late spermatids, and this is correlated with a delay in sperm release. However, it has not been shown if disruption of cortactin expression has similar effects on TBCs associated with the basal junction complexes and whether or not this has an effect on junction removal. Here we use electron microscopy (EM) and stimulated emission depletion (STED) super resolution microscopy to evaluate the phenotype of rat testes injected 5 times over a 9‐day period with siRNA targeting cortactin, and use western blots to confirm the knockdown. In vivo knockdown of cortactin in rat testes disrupts basal TBCs when sections labeled for ORP9 and cortactin/actin are evaluated by STED, and leads to the presence of redundant junction related material (ectoplasmic specializations) when evaluated by EM. These data are consistent with the conclusion that TBCs internalize basal junctions and are likely part of the mechanism of spermatocyte translocation from basal to adluminal compartments of the epithelium.

Support or Funding Information

Supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to A Wayne Vogl (RGPIN‐2018‐03727).
CRP and Cra have Antagonistic Roles in the Regulation of the Fructose Operon in Vibrio cholerae
Authors: Beck Christina , Perry Sayde , Liu Jane
Published: April 2020

Vibrio cholerae, the causative agent of cholera, survives in both the human small intestine, where pathogenesis occurs, and aquatic reservoirs by adapting to changes in carbon source availability. In order to metabolize fructose, one of the most prevalent sugars in freshwater environments, V. cholerae must selectively produce the necessary metabolic machinery. Here, in studying how V. cholerae regulates fructose metabolism at the genetic level, we found that Cra (catabolite repressor/activator) and CRP (cAMP receptor protein), two well‐described transcriptional regulators in Escherichia coli, have antagonistic roles in regulating the expression of fruB, a member of the fructose operon fruBKA. Using transcriptional assays, qRT‐PCR, and western blot analysis to evaluate changes in gene expression, we found Cra to be a repressor of fruB and CRP to be an activator of fruB. We also found CRP to be a repressor of Cra, suggesting that CRP and Cra interact to affect downstream fruB expression. In order to further elucidate the mechanisms by which Cra and CRP regulate fruB expression, we are currently focusing our efforts on mapping the fruB promoter region. Although more remains to be understood about the relationship between the two regulators, this work has elucidated previously unknown roles of Cra and CRP in regulating fructose metabolism at the genetic level in this facultative pathogen.

Support or Funding Information

National Institutes of Health, The Camille and Henry Dreyfus Foundation
Silk fibroin converts quiescent cardiomyocytes to pacemaker cells
Authors: HU YU-FENG
Published: April 2020

Introduction

Biological pacemakers, generated by cell or gene delivery, have been developed as hardware‐free alternatives to electronic devices. However, the challenges associated with gene manipulation and immunogenicity complicate clinical applications.

Hypothesis

The biological pacemakers could be generated by the biomaterial, a gene and vector‐free strategy.

Methods

Key features of pacemaker cells were assessed by patch clamp and calcium image in the rodent ventricular cardiomyocytes (VMs) after exposure to silk fibroin (SF) to characterize the conversion to pacemaker cells. The optical mapping was used to map in‐vivo biological pacemaker activity from SF‐implantation site. We used whole transcriptome analysis to explore the underlying mechanisms.

Results

SF‐induced pacemaker cells exhibited both functional and morphological features of genuine sinoatrial node (SAN) cardiomyocytes in vitro including long and slender morphology, HCN4 and Cx45 expressions, membrane (increased If and IK1 current) and calcium clock (diastolic calcium release). When implanted into the rat ventricle, SF induced biological pacemaker activity which functioned as a surrogate in situ SAN. Whole transcriptome analysis showed SF induced ectopic expression of vascular endothelial cadherin (Cdh5) on quiescent VMs. Cdh5 expression activated β‐catenin signaling and drove simultaneous expression of transcription factors during SAN embryogenesis, which converted quiescent VMs into pacemaker cells.

Conclusions

The SF‐based approach represented a new strategy to generate pacemaker cells for future clinical applications.

Support or Funding Information

NHRI‐EX108‐10513SC
Suppression of histamine H1 receptor gene expression by antihistamines with inverse agonist activities
Authors: Fukui Hiroyuki , Mizuguchi Hiroyuki , Kitamura Yoshiaki , Takeda Noriaki
Published: April 2020

Allergic rhinitis is an incurable disease whose prevalence rate is over 30% in Japan. Antihistamines are first‐choice drugs for the therapy of the disease. However, sole treatment of antihistamine is not enough to obtain satisfactory control. We elucidated histamine‐induced histamine H1 receptor gene expression as a pathological mechanism of allergic rhinitis. Treatment of epinastine, an antihistamine, to allergic rhinitis patients showed alleviation of nasal symptoms and correlated suppression of histamine H1 receptor mRNA elevation in nasal mucosa. We observed inverse agonist activities in some antihistamines. Suppression of inositol phosphates accumulation and suppression of histamine H1 receptor gene expression showed good correlation. These results suggested that antihistamines with inverse agonist activities have advantage for the therapy of allergic rhinitis.

Support or Funding Information

Research Grant from The Osaka Medical Research Foundation for Intractable Diseases
3D Visualization of Depression and Anxiety
Authors: Stalets Marissa , Rodgers Emily , Dufeau David
Published: April 2020

We present a novel and technologically enhanced teaching tool geared towards both patients, students, and clinicians with the primary goal of educating these groups about the anatomical and biochemical implications of various syndromes involving depression and anxiety. Our secondary goal is to inform patients, students, and clinicians about the most common treatment methods used to treat these syndromes. We achieved this by mapping the mesolimbic system, otherwise known as the “reward pathway,” and other relevant neuroanatomical structures using a high‐resolution cryosection dataset made available by the Big Brain project at McGill University and constructing 3D visualized models using 3D analysis software tools. The results of this project were compiled into a series of three interactive educational modules serving each of our goals. The first module described the neuroanatomical structures involved in syndromes of depression and anxiety along with their functions. The second module describes various forms of clinical depression and treatment options, including the mechanism of action and how it applies to the underlying disease process. The third module did the same but described anxiety rather than depression. This tool is valuable to patients and care providers alike because it explains neuroanatomical and biochemical aspects of these syndromes in a straight‐forward and easy‐to‐understand visual manner. It also describes the context of the best pharmacological interventions for each syndrome, which is a substantial step to help engage patients in their personalized mental health treatment plan.

Support or Funding Information

Thank you to Dr. David Dufeau and Marian University College of Osteopathic Medicine for the resources and guidance throughout this research project.
Atrial Natriuretic Peptide Prevented Lipid‐Induced Kidney Injuries by Inhibiting Endoplasmic Reticulum Stress
Authors: ZHAO XIAODUO , Kong Yonglun , Li Suchun , Liang Baien , Li Chunling , Wang Weidong
Published: April 2020

Hyperlipidemia plays an important role in the progression of kidney injury, likely due to renal lipotoxicity. The purpose of the present study was to investigate whether atrial natriuretic peptide (ANP) prevented lipid‐induced injury through inhibiting endoplasmic reticulum (ER) stress and apoptosis in the kidney. In human proximal tubule HK2 cells, saturated fatty acid palmitic acid (PA) treatment (0.4mM) for 24h markedly increased protein abundance of two ER stress makers BiP (3.7‐folds of controls) and CHOP (5.2‐folds of controls), which was associated with upregulated cleaved‐caspase 3 expression (3.2‐folds of controls) and decreased ratio of Bcl2/Bax (0.76‐folds of controls), two markers of apoptosis. ANP treatment (10pM) markedly decreased protein abundance of BiP (2.8‐folds of controls), CHOP (3.7‐folds of controls), and cleaved‐caspase 3 (1.7‐folds of controls), and increased the ratio of Bcl2/Bax (1.2‐folds of controls) in HK2 cells treated by PA. Co‐treatment with ANP and LBQ657, a neprilysin inhibitor preventing hydrolysis of ANP, showed better protective effects than ANP treatment alone in HK2 cells treated with PA. ANP is known to stimulate intracellular cGMP‐PKG signaling pathway, the role of cGMP and PKG in ANP‐induced protection after PA treatment in HK2 cells was thus investigated. Sildenafil, an inhibitor of phosphodiesterase‐5 (PDE5), and exogenous 8‐Br‐cGMP markedly suppressed PA‐induced ER stress and apoptosis, whereas KT‐5823, a selective cGMP‐dependent PKG inhibitor, blocked protection of ANP or cGMP in HK2 cells treated with PA. A synthesized, specific PDE5 inhibitor LW1646 showed slightly better prevention than sildenafil on PA‐induced HK2 cell injury. ANP attenuated PA‐induced upregulation of inositol 1,4,5‐triphate receptor (IP3R) that is involved in calcium efflux from ER to cytoplasm. ANP decreased PA‐induced intracellular calcium oscillation, prevented PA‐induced mitochondrial depolarization, and promoted mitochondrial biogenesis in HK2 cells. Compared with controls, in mice fed with high‐fat diet for 8wk, the protein expression of BiP and CHOP in the kidney cortex showed 2.8‐fold and 2.3‐fold increases, respectively, which was markedly prevented by sildenafil (15mg/kg·BW/day, gavage). In conclusion, these findings demonstrated that ANP inhibited lipid‐induced ER stress and apoptosis, likely via activating the cGMP‐PKG signaling pathway and maintaining mitochondrial function, it may be a potentially therapeutic target for lipid‐induced kidney injury.
Cannabidiol Effects on Post‐Seizure Neurogenesis
Authors: Victor Tanya R. , Elmes Matthew W. , Deutsch Dale G. , Tsirka Stella E.
Published: April 2020

Epilepsy is a complex disorder characterized by a propensity to generate repetitive seizures with molecular and cognitive consequences. Many pharmacological treatments for epilepsy target neurotransmitters or ion channels, leaving nearly 30% of patients resistant to current medications. The recent approval of cannabidiol (CBD), a non‐psychotropic component of Cannabis sativa, has demonstrated the ability to lessen seizure severity and curb neuroinflammation in experimentally induced seizures in animal models. While CBD has been shown to effect multiple pathways and targets, its complete mechanism of action is still not understood.

Current models of induced seizures mostly utilize treatments before or during epileptic events, though this practice does not normally occur in the clinic. Here we utilize a more translational model where CBD treatment is administered after the first seizure event. An inciting seizure increases the likelihood of secondary seizures due to epileptogenesis, where seizure susceptibility is increased due to molecular and structural changes within the central nervous system (CNS) parenchyma. The epileptogenic process includes changes in axonal sprouting, cell death, neurotransmitter release, and in the hippocampus aberrant granule neural network reorganization and neurogenesis. Microglia, the resident immune cells in the brain, are involved in hippocampal neurogenesis, controlling apoptosis of the newborn neurons and ultimately the numbers of granule cells. The objective of this study is to investigate whether CBD modulates microglial involvement in neurogenesis post seizure.

Methods

Following seizure induction in 8–10 week old mice, mice are given BrdU for 7 days post seizure to monitor cell proliferation and survival. Newly generated neurons are assessed using NeuN and DCX, to identify differentiated neuroblasts and mature neurons, respectively. BrdU+ cells are used for quantification and assessment of differentiation and survival of new cells in the dentate gyrus. Neural stem cell (NSC) levels in the hippocampus are also compared using SOX2/GFAP co‐staining.

Results

Our data suggest that CBD affects the number of new neurons after seizure. Mechanisms contributing to neurogenesis will be explored.

Conclusions

CBD contributes to microglia‐mediated changes in the numbers of granule cells and neurogenesis in a model of epilepsy.

Support or Funding Information

This work was partially supported by an NSF Predoctoral Fellowship and SBMS funds to TRV.
Novel Effect of Chitooligosaccharide on Cyst Growth Retardation in an in vitro Cyst Model of Polycystic Kidney Disease
Authors: Pathomthongtaweechai Nutthapoom , Muanprasat Chatchai
Published: April 2020

Polycystic kidney disease (PKD) is a common inherited disorder characterized by multiple fluid‐filled cysts that replace normal renal parenchyma along the renal tubules leading to impaired renal function and end‐stage renal disease (ESRD). It has recently been reported that the activation of AMP‐activated protein kinase (AMPK) by metformin inhibits mammalian target of rapamycin (mTOR)‐mediated cell proliferation and cystic fibrosis transmembrane conductance regulator (CFTR)‐mediated chloride secretion in Madin‐Darby canine kidney (MDCK) cell, a cell line derived from distal nephron of dog kidneys. Therefore, AMPK activators are proposed to be a promising agent for PKD treatment. Recently, our research group has shown that chitooligosaccharide (COS) derived from chitin/chitosan activates AMPK in intestinal epithelial cells. This study was conducted based on the hypothesis that AMPK activation by COS may inhibit cyst growth in the in vitro cyst model of PKD. We found that COS at the concentrations of 10, 50, and 100 μg/mL decreased cyst growth in a concentration‐dependent manner without toxicity measured by MTT assays. Immunoblotting analysis showed that COS at 100 μg/mL activated AMPK and this effect was abolished by STO‐609, a calcium/calmodulin‐dependent kinase kinase beta (CaMKKβ) inhibitor. Moreover, COS could elevate the level of intracellular calcium. These results suggest that COS inhibits cyst progression by activation of AMPK and the increment of intracellular calcium level via CaMKKβ. Therefore, COS has novel potential application for the treatment of polycystic kidney disease.

Support or Funding Information

Faculty of Medicine Ramathibodi Hospital, Mahidol University
Size Matters! Evaluating the Effect of Model Size on Anatomy Learning
Authors: Yang Jack (Xiaozhi) , DeYoung Veronica , Xue Yuanxin , Nehru Amit , Hildebrand Alexandra , Brewer-Deluce Danielle , Wainman Bruce
Published: April 2020

Historically, learning anatomical specimens was limited to studying cadaveric materials, and by extension, specimens which are “life‐sized”. Recent technological advancements in 3D scanning and printing now allow for the production of inexpensive, durable anatomical replicas at virtually any size. This, however, creates a dilemma: what is the most effective model size to learn from? The goal of this project is to discover the appropriate size of an object to learn nominal anatomy and thus provide a critical step in improving anatomic education.

We hypothesize that there is a curvilinear relationship between model size and learning, where a model too small or too big would not be the most conducive to learning and an ideal intermediate size can be determined. In this study, undergraduate students (n = 351) without prior anatomical training learned from four bones of varying normal anatomical size and features and were assessed on their ability to identify various landmarks. Thoracic vertebra (VE), hemipelvis (HE), sphenoid (SP), and scapula (SC) was 3D‐printed at four different scalar sizes. The VE and HE models were printed at 50%, 100%, 200%, and 400% scale, while SP and SC models were printed at 50%, 100%, 200%, and 300% scale. Each participant was randomly assigned to a group of two bone models (VE/HE or SP/SC) of a certain size, and randomized across the order in which they learned the models. They were then tested on the respective real bone specimens, followed by a qualitative survey reporting their experience with the 3D‐printed models, a Mental Rotations Test (MRT), and an Operation Span Test (OSPAN). Data collection for the 50% SP/SC group is still ongoing.

Multiple regression suggested significant effects of Model Type, Model Size, MRT and OSPAN, (F(9, 596) = 17.96, p = 0.000, R2 = 0.2133). The most significant predictor of test score was MRT, which suggested a 10% increase in MRT score is associated with a ~3% increase in test score. The score variability independently accounted for by MRT and OSPAN was 14.6%, while the variability independently accounted for by model size and type was 7.8%. This means, that while test scores are primarily driven by participants’ mental rotation ability, model size remains an important feature that can be manipulated to improve learning. 3D printing allows for this in a cost‐effective way.

Support or Funding Information

This study was funded by the Education Program in Anatomy at McMaster University. Many thanks to the University of Buffalo for 3D printing the 400% VE and HE models.