bluuebunny/crossref_metadata_2025 huggingface dataset:
Codeine Asymmetrically Inhibits The Laryngeal Adductor Reflex
Authors: Musselwhite M. Nicholas , Shen Tabitha Y. , Rose Melanie J. , Olsen Wendy L. , Pitts Teresa G. , Bolser Donald C.
Published: April 2020

The laryngeal adductor reflex (LAR) is a fundamental airway protective behavior that prevents penetration of material from the upper airway into the trachea. Aspiration risk increases in humans after a single dose of an opioid and pneumonia is a common occurrence in opioid users. The LAR consists of a short latency (~10 ms), short duration (~10 ms) burst (R1) and a longer latency (~40ms) burst (R2) in laryngeal electromyogram (EMG) activity. The R1 LAR is thought to be primarily a brainstem reflex. We hypothesized that systemic administration of codeine, one of the most commonly prescribed opioids, would suppress the R1 LAR in an animal model that robustly expresses airway protective behaviors. The LAR was elicited in anesthetized, spontaneously breathing cats (n=13) by electrical stimulation (2 Hz, 0.1 ms pulse duration) of the superior laryngeal nerve (SLN) or mechanical stimulation of the vocal folds with a von Frey filament. Bilateral thyroarytenoid (TA) and midline posterior cricoarytenoid (PCA) muscle electromyograms (EMGs) were recorded. The R1 LAR manifested as a short latency (approx. 10 ms) evoked response consisting of peaks and troughs in both the TA and the PCA EMGs. Codeine (0.1–10 mg/kg, iv) dose dependently inhibited the magnitude of the contralateral TA and PCA LAR by approximately 60% but had no significant effect on the LAR in the ispilateral TA EMG. Similar lateralization of the effect of codeine was observed in animals in which the LAR was elicited by mechanical stimulation of the vocal folds. These effects were similar in animals whether only one or both SLNs were sectioned. In separate animals, the codeine dose response was conducted after pretreatment with the NMDA antagonist, MK‐801 (10 μg/kg) via the vertebral artery. MK‐801 enabled depression of the TA LAR on the ipsilateral side by iv codeine, but did not enhance the reduction in the contralateral TA or PCA LAR relative to animals that only received this opioid. These findings support lateralized suppression of the LAR by opioids that is mediated by central NMDA receptors. The differing effects of codeine on the LAR in the PCA and ipsilateral TA EMGs suggest that opioids may disrupt laryngeal protective actions through asymmetric positioning of the vocal folds.

Support or Funding Information

Supported by NIH HL131716 and 3OT2OD023854
X‐ray crystal structure of MonCI, an epoxidase from the monensin biosynthesis pathway
Authors: Wang Qian , Nitka Tadeusz , Gao Qin , Lee Wen-Yee , Chen Xi , Irimpan Mathews , Vukovic Lela , Kim Chu-Young
Published: April 2020

Polycyclic polyether natural products have gathered much attention due to their useful therapeutic properties including antimicrobial, antifungal, and anticancer activities. All polyether natural products contain multiple cyclic ether groups but they vary in the number, size, and arrangement of the cyclic ether groups. The ether rings found in natural polyethers are formed via enzymatic epoxidation and epoxide ring‐opening reactions. We have determined the 1.9 Å resolution crystal structure of MonCI, the flavin‐dependent epoxidase responsible for biosynthesis of the ionophore polyether antibiotic monensin. Our structural and molecular dynamics simulation studies indicate that the shape of the substrate‐binding pocket in MonCI dictates the stereoselectivity of epoxidation.

Support or Funding Information

The University of Texas System STARs Award
Investigating Sex Hormone Influence on Nesfatin‐1 Signaling through qPCR
Authors: Schnell Abigayle Lyn , Samson Willis K. , Yosten Gina L.C.
Published: April 2020

Nesfatin‐1 is a potent anorectic peptide that participates in the hypothalamo‐pituitary‐adrenal (HPA) axis, and previous research indicates that the expression and function of nesfatin‐1 is influenced by female sex hormones. While nesfatin‐1 signaling in the HPA axis of male animals is well‐studied, nesfatin‐1 signaling in the HPA axis of female animals remains to be investigated. Changes in nesfatin‐1 action and expression in dispersed rat pituitaries as well as hypothalamus, pituitary, and adrenal gland tissues harvested across the estrus cycle was determined through use of qPCR. However, housekeeping genes used for normalization of qPCR data have not been validated for use in studies examining the role of female sex hormones. Therefore, tissues were collected from cycling female rats to perform qPCR experiments measuring expression of the most commonly used housekeeping genes in order to determine which housekeeping genes are stable across the estrus cycle. These studies suggest that nesfatin‐1 expression and signaling in the HPA is influenced by female sex hormones. Furthermore, our data indicate which housekeeping genes are most appropriate for research on sex differences.

Support or Funding Information

National Institute of Health
Regulation of glioblastoma tumor growth and stem cell properties through Gα12: an upstream player in YAP signaling
Authors: Chaim Olga M. , Miki Shunichiro , Ma Jianhui , Flores Anabel , Miyamoto Shigeki , Furnari Frank , Brown Joan Heller
Published: April 2020

Glioblastoma multiforme (GBM), the most common and aggressive form of brain tumor, has been associated with the dysregulation of G‐protein coupled receptor signaling. Data from the Cancer Genome Atlas demonstrate remarkable upregulation of mRNA for the Gα12 protein, which is elevated in 26% of GBM patient tumors (the highest rate for all tumor samples surveyed). The Gα12 protein couples GPCRs to guanine nucleotide exchange factors (GEFs) for RhoA and was originally isolated as an oncogene. Furthermore, the subset of GPCRs that couples to Gα12 to activate RhoA includes S1P, LPA, and thrombin, inflammatory mediators that would be generated in the tumor cell environment. GPCR signaling to RhoA leads to activation of the transcriptional co‐activator YAP (Yes‐associated protein 1 gene). The role of YAP in growth of glioblastoma cells and in vivo GBM tumor development was explored in our previous studies (Yu et al. Oncogene 7(41): 5492–5507, 2018) using human glioblastoma cell lines and glioma stem cells (GSCs) derived from patient derived xenografts (PDX). We showed that shRNA mediated knockdown of YAP in GSC‐23 PDX cells significantly attenuated in vitro self‐renewal capability assessed by limiting dilution, oncogene expression, neurosphere formation, and stem cell gene expression. Orthotopic xenografts of the YAP knockdown PDX cells formed significantly smaller tumors and were of lower morbidity than wild‐type cells. Our current work examines the effects of Gα12, which would transduce effects of known and orphan GPCRs upstream that might be activated in the tumor environment to RhoA. We knocked down Gα12 using shRNA in two GBM cancer stem cell lines, GSC‐23 and HK‐281, decreasing Gα12 mRNA levels by 80% without compensatory upregulation in the expression of the homologous Gα13 protein. Loss of Gα12 attenuated GSC‐23 stemness as determined by limiting dilution assay and reduced mRNA levels for canonical stem cells genes (CCND1, NANONG, OCT4, SOX2 and NESTIN) by 35% to 60%. We are currently examining growth of GSC‐23 control and Gα12 knockdown cells following orthotopic injection into mouse brain. Histopathological findings revealed that the deletion of Gα12 reduced tumor‐derived cell migration and invasiveness. Gα12 knockdown tumors also presented with increased areas of necrosis. In vitro transwell assays confirmed that Gα12 knockdown reduced agonist‐stimulated GSC‐23 cell migration. We are exploring the role of YAP‐dependent genes (e.g. Thbs‐1, Dusp‐1, and CCN1) which were selectively decreased in tumors from PDX cells lacking Gα12 to determine their role in tumor cell migration and survival.

Support or Funding Information

NIH grant CA218859
Tibetans resident at intermediate altitude (1300 m, 4327 ft) show similar hypoxic ventilatory responses but blunted heart rate responses to poikilocapnic hypoxia
Authors: Moya Esteban A. , Yu James J. , Brown Spencer , Gu Wanjun A. , Lawrence Elijah A. , Carlson Ryan , Brandes Anna , Wegeng William A. , Amann Kassaundra , McIntosh Scott , Powell Frank L. , Simonson Tatum S.
Published: April 2020

Population living at high altitudes for hundreds of generations exhibit distinct respiratory and cardiovascular responses to hypoxia relative to other populations. The extent of these differences at intermediate altitude is less understood and could provide important insight into ancestry specific physiological responses in the absence of hypoxia. We hypothesized that Tibetans living at intermediate altitude (1300 m, 4327 ft) exhibit an increased hypoxic ventilatory response (HVR) and an elevated hypoxic heart rate response (HHR) compared to Han Chinese examined at the same altitude. To estimate O2 sensitivity, we measured ventilation (VI), heart rate (HR) and O2 saturation (Sat) under hyperoxic conditions (30% O2) and then during a hypoxic stimulus (10% minimum desaturation) keeping end‐tidal CO2 (EtCO2) levels constant (isocapnia) or allowing changes in the EtCO2 (poikilocapnia). We quantified HVR and HHR as change in VI and HR between hyperoxia and hypoxia standardized per the change in O2 Sat (ΔVI/ΔSat and ΔHR/ΔSat respectively). To estimate CO2 sensitivity, we measured changes in VI and HR responses between poikilocapnic and isocapnic hypoxia and standardized these changes per change in EtCO2 (ΔVI/ΔEtCO2 and ΔHR/ΔEtCO2 respectively). We did not find significant differences in ΔVI/ΔSat and ΔVI/ΔEtCO2 between Tibetan and Han Chinese during isocapnic and poikilocapnic hypoxia. The ΔHR/ΔSat between populations during isocapnic hypoxia was also not significantly different. However, Tibetans exhibited a blunted ΔHR/ΔSat during poikilocapnic hypoxia compared to Han Chinese (35.8% decrease, p < 0.02) with a significant ancestry effect in women (37.6% decrease, p < 0.009). The mean value of ΔHR/ΔEtCO2 was positive in the Han Chinese group but negative in Tibetan (169% difference, p < 0.02), indicating that Tibetans had a blunted HR response when CO2 is not controlled. Our results show that HVR responses in Tibetan individuals with high‐altitude ancestry is not different than Han Chinese residents at comparable intermediate altitude, but the HHR to hypoxia during poikilocapnia is blunted in individuals with Tibetan ancestry. These results suggest that, at intermediate altitude, individuals with Tibetan ancestry exhibit an attenuated heart rate response to hypoxia due to an increased contribution of the CO2 chemosensory response.

Support or Funding Information

Supported by NIH 1RO1HL145470 and R01 HL‐081823.
The Big Q: Is Q‐methodology valid for evaluating a large‐scale, cross‐disciplinary anatomy and physiology course?
Authors: Saraco Anthony N. , Brewer-Deluce Danielle , Akhtar-Danesh Noori , Wainman Bruce C.
Published: April 2020

Introduction

Course evaluations are an important tool to gather feedback on the structure of a course, instructor effectiveness, and the overall learning experience. Critically, the Likert scale approach used by most institutions lacks course specificity and the difference between responses cannot be assumed equal (eg. “strongly agree – agree” vs. “agree – neutral”). This makes evaluating the effectiveness of a course and identifying areas that need improvement difficult. Q‐methodology is a technique that mitigates these issues by polling students for qualitative feedback statements that represent prevalent opinions of the course, then asking them to rank the statements relative to each other. Students are then clustered by shared opinions, values, and preferences.

Methods

This study uses Q‐methodology to assess student opinions on an undergraduate anatomy and physiology course (850 students). Specifically, students across five disciplines (midwifery, bachelor of health sciences, engineering, nursing, and integrated biomedical sciences) enrolled in the same second‐year undergraduate anatomy and physiology course were recruited into the study. All students experienced the same lecture and laboratory components as well as discipline‐specific tutorials. Students were asked to rank 37 statements relative to each other using an online platform. A by‐person factor analysis was completed using the qfactor program in Stata. Overall, the goal of this study was to validate Q‐methodology as an assessment modality across different populations experiencing the same course.

Results

143 students participated in the study (70.6% female, 25.2% male, 4.2% rather not specify; median age: 19, range: 18 – 38). The by‐person factor analysis classified students into three significantly different groups (22 students unassigned) representing 1) students who greatly appreciated the use of cadaveric specimens (n = 55), 2) students who were extremely dissatisfied by the means of evaluation (n = 40), and finally 3) students who despised the virtual reality (VR) supplementary resource (n = 26). Group 1 expected a significantly higher grade than the other two groups (p<0.05). No demographic data correlated with the groups, nor did discipline. All three groups agreed upon six consensus statements.

Conclusion

Critically, this study uncovered three distinct opinion patterns spanning all five academic disciplines within the course. The study provided guidance for course reform and suggested that discipline does not predict course evaluations. The study supports the use of Q‐methodology analyses for assessing student opinions on a large scale. Future work looks to re‐assess student course evaluations in the same course to determine how Q‐methodology outcomes change in response to “Q”‐directed course reform.
Concerted diurnal rhythms of gut microbiota with salt‐sensitive hypertension and renal inflammation
Authors: Mandal Juthika , Chakraborty Saroj , Cheng Xi , Galla Sarah , Hindupur Anay , Saha Piu , Yeoh Beng San , Mell Blair , Yeo Ji-Youn , Kumar Matam Vijay , Yang Tao , Joe Bina
Published: April 2020

Aberrant diurnal rhythms of Blood Pressure (BP) are well known to be associated with hypertension, however, whether rhythmicity of microbiotal composition is associated with salt sensitive hypertension remains unknown. We hypothesized that BP and gut microbiotal composition follow synchronous rhythms in high salt fed Dahl Salt Salt‐Sensitive (S) rats and contribute to the progression of hypertension. To test this hypothesis, we examined groups of Dahl S rats for their diurnal rhythms of both the gut microbiota and blood pressure in low salt (0.3%) and high salt (2%) conditions. In high salt fed animals, Firmicutes/Bacteroidetes (F/B) ratio significantly changed between the dark (active) and light (rest) phases, which correlated with the diurnal rhythmicity of BP suggesting salt mediated rhythmic regulation of microbiota. Diurnal rhythms of Firmicutes, Bacteroidetes and Actinobacteria were independently associated with BP and significant in high salt fed animals. Discrete genera were observed to correlate independently or interactively with one or more of the following 3 factors‐1) BP rhythm, 2) dietary salt, 3) amplitude of BP. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUST) analysis revealed diurnal rhythmicity of microbiotal pathways. During the active phase of the host, microbiota upregulated biosynthetic processes whereas during the resting phase of the host, microbiota upregulated degradation pathways of metabolites. These diurnal changes in microbiota, their functional pathways and BP response were prominently associated with a concerted rhythmicity of renal Lipocalin 2 and Endothelin1. Collectively, these data demonstrated the existence of synchronous diurnal rhythms of BP and renal inflammation with diurnal reshaping of gut microbiota in salt‐sensitive hypertension. Such a concerted rhythmicity with peaks observed at the mid‐active phase suggests that targeting this timepoint to reshape microbiota and/or intervene with medication could benefit hypertensives.

Support or Funding Information

National Institutes of Health (R01HL143082)
Targeting white matter microgliosis using minocycline in a co‐morbid preclinical rat model of Alzheimer’s disease and stroke
Authors: Agapova Victoria , Blanchet Victoria Helene , Allman Brian , Whitehead Shawn
Published: April 2020

Current literature indicates a potential synergistic interaction between stroke and Alzheimer’s disease (AD). White matter (WM) inflammation, which is commonly triggered by cerebrovascular disease and AD, disrupts the normal function of white matter; and can trigger and exacerbate dementia‐type symptoms such as memory loss and executive dysfunction. Recent work in our lab demonstrated, for the first time, a link between stroke, AD‐related pathology, and WM inflammation. A unilateral lacunar stroke induction in a comorbid rat model of AD and stroke resulted in microglial activation within the WM tracts and subsequent impairment in behavioural flexibility, an important component of executive function. As such, the aim of the current study was to evaluate the effect of microglia targeting on WM pathology, executive function, learning, and memory in the co‐morbid rat model of AD (TgAPP21) and stroke. Minocycline, a tetracycline‐class antibiotic and an anti‐inflammatory drug, has been shown to decrease apoptosis, inflammation, and microglial activation following cerebrovascular injury. To study the behavioural outcomes of minocycline treatment, TgAPP21 rats received an endothelin‐1‐induced subcortical stroke at the age of 8 months. Following stroke, animals received intraperitoneal injections of minocycline or saline twice daily for four days to supress microglial activation. Cognitive behavioural flexibility post‐stroke was analyzed using set‐shifting operant conditioning test, while spatial learning and memory were assessed using Morris water maze. Immunohistochemistry was performed to evaluate the extent of microglial activation by detection of OX‐6 positive microglia in corpus callosum, internal capsule, and hippocampus. Minocycline treatment significantly decreased the number of errors committed by TgAPP21 rats on the visual cue component of the set‐shifting test, suggesting improved learning of the original visual cue task. However, minocycline also impaired animals’ ability to undergo an extra‐dimensional strategy shift from the original visual cue to a new spatial cue task, suggesting impaired behavioural flexibility. Further, minocycline treatment showed no significant effect on learning and memory of the Morris water maze task in TgAPP21 rats. Collectively, the data indicate that minocycline has distinct effects on the learning component of set‐shifting and Morris water maze tests, though further histological evaluation of OX‐6 positive microglia within WM tracts and hippocampus will contextualize current findings.

Support or Funding Information

Natural Sciences and Engineering Research Council (NSERC), Canadian Institutes of Health Research (CIHR), Canadian Foundation for Innovation (CFI), Canadian Consortium for Neurodegeneration and Aging (CCNA)
Regulation of the E3 Ligase WWP2 by GPCR Activation and CDH1 Mediates ALIXdependent Lysosomal Sorting
Authors: Shahid Shafaq , Kotsis Christopher , Singh Harjoyt , Dores Michael Robert
Published: April 2020

G protein‐coupled receptors (GPCRs) comprise the largest and most diverse family of membrane receptors. Dysregulated GPCR signaling is linked to multiple diseases, including inflammation, vascular disease, and many cancers. Attenuation of GPCR signaling is mediated by internalization and lysosomal degradation. GPCRs signal to their endocytic sorting machinery to initiate trafficking at the plasma membrane as well as at the limiting membrane of early and late endosomes. GPCRs like protease‐activated receptor 1 (PAR1) can stimulate the ubiquitination and activation of ALIX, an adaptor protein that sorts GPCRs with intracellular YPXnL motifs into budding intraluminal vesicles at multivesicular endosomes. This process is mediated by the NEDD4‐family E3 ubiquitin ligase WWP2. Here, we show that stimulation of multiple YPXnL‐motif receptors, including PAR1, the purinergic receptor P2Y1 and the histamine H2 receptor, induce ubiquitination of WWP2 during receptor lysosomal sorting. In addition, we have identified a novel role for the E3 ligase CDH1 in modulating WWP2 ubiquitination. Transient depletion of CDH1 leads to elevated WWP2 ubiquitination and receptor downregulation, suggesting a potential mechanism for conditionally modulating WWP2 activity during receptor trafficking. Our current research aims to elucidate the common connection between the signaling pathways activated by YPXnL GPCRs and the regulation of CDH1 and WWP2.
The Powerhouse of Depression: The Impact of MTCH‐1 Deficiency on Reward Signaling in C. Elegans
Authors: Fong Megan J. , Langner Grace J. , Jazari Malianna Bahar , Sakiyama Matthew S.
Published: April 2020

Major Depressive Disorder (MDD) is the leading cause of disability worldwide. One of MDD’s most troubling symptoms is the loss of reward firing, or anhedonia. Current publications indicate that MTCH‐2, a gene involved with mitochondrial transport, is related to a decrease in firing rates in the hippocampus of mice. Additionally, a GWAS reported an association between MTCH‐2 and neuroticism in humans. Consequently, we hypothesize that a MTCH‐2 related decrease in firing rates in the nucleus accumbens could potentially play a role in the development of depressive symptoms.

The C. elegans MTCH‐1 protein shows an 87% homology to the human MTCH‐2 protein. Here, we examine the relationship between the MTCH‐1 gene, a previously unexamined gene, and C. Elegans reward firing. Because C. elegans reward firing levels have been associated with varying speed change fluidity in past studies, we measure C. elegans motility as a proxy for reward firing.

In this study, we compare motility and gene expression between MTCH‐1 deficient worms and wildtype worms. We are using WormTracker to quantify motility based upon turn count in videos of worms recorded on ToupCam. Post all motility tests, a PCR and GFP assay on worms serve to verify their phenotypes. After our assays are concluded, we plan to use ANOVA for statistical analysis of our results. Thus, together, these data may demonstrate the influence of MTCH‐2 on reward firing, providing new insights into indicators and treatments of depression.

Support or Funding Information

This work was supported and funded by the Nueva School. The Shen Lab at Stanford University provided lab equipment and N2 worms. Worms and technical support were provided by Caenorhabditis Genetics Center (CGC) ‐ College of Biological Sciences.
Dysregulation of Skeletal Muscle Mitochondrial Function following Critical Illness: a Translational Approach
Authors: Gill Luther , Simon Liz , Molina Patricia
Published: April 2020

Bioenergetic failure is a significant clinical consequence of sepsis‐induced critical illness and is characterized by reduced skeletal muscle mitochondrial density, and mitochondrial dysfunction. Early activation of mitochondrial biogenesis is associated with survival and improved physical function in critically ill patients. Therefore, investigating mechanisms associated with early activation of mitochondrial biogenesis may improve targeting of future rehabilitative strategies. A rodent cecal‐ligation and puncture (CLP) model was used to mimic sepsis‐induced critical illness. The objective of the study was to determine whether CLP impaired genes implicated in mitochondrial function in the diaphragm. The CLP model involved ligation of 50% of the cecum below the ileocecal valve in adult C57BL6 mice, followed by needle puncture of the cecum resulting in mid‐grade sepsis. Mice survived for 48 hours or more, following injury. Diaphragm and limb muscles were harvested 24 hours following CLP (N=6) and following a sham CLP procedure (N=6). Gene expression of mef2c, myh1, and pgc1‐α, were significantly decreased in the diaphragm of CLP injured animals when compared to controls. In addition, ubiquitin ligases, murf1 and atrogin were increased in the diaphragm 24 hours after injury (p< 0.01). Our results indicate that sepsis‐induced critical illness significantly impacts the expression of genes implicated in mitochondrial homeostasis and atrophy. Ongoing studies will identify whether CLP injury decreases skeletal muscle mitochondrial function.

Support or Funding Information

NIH grants U54 GM104940 and Department of Physiology Funds.
Contribution of NLRP3 Inflammasome Activation to Glomerular Injury during Hyperhomocysteinemia with and without Enhanced Exosome Secretion
Authors: Huang Dandan , Li Guangbi , Camus Sarah , Li Ningjun , Ritter Joseph K. , Li Pin-Lan
Published: April 2020

Hyperhomocysteinemia (hHcy) has been demonstrated to activate NLRP3 inflammasome leading to podocyte injury and glomerular sclerosis. However, it remains unknown whether exosome‐mediated secretion of NLRP3 inflammasome products is involved in the development of glomerular injury during hHcy. In the present study, we examined the possible role of increased exosome secretion during NLRP3 activation in the inflammatory response and tested whether without robust release of exosomes, inflammatory response and glomerular injury still occur. Exosome biogenesis inhibitor‐GW4869, lysosome trafficking stimulator‐rapamycin or ASM inhibitor‐amitriptyline were used in mice to answer this question. By nanoparticle tracking analysis, it was found that hHcy significantly increased urinary exosome release in WT/WT mice. In Smpd1trg/Podocre (podocyte‐specific ASM transgenic) mice, this hHcy‐induced increase in urinary exosomes was significantly enhanced. Pretreatments of WT/WT mice and Smpd1trg/Podocre mice with amitriptyline (Ami), GW4869 (GW) and rapamycin (Rap) all prevented increases in urinary exosomes during hHcy. In podocytes isolated from WT/WT mice and Smpd1trg/Podocre mice, similar results were obtained when we measured exosomes released from these cells after different treatments. Confocal microscopy demonstrated that Ami not only prevented hHcy‐induced NLRP3 inflammasome formation and activation in glomeruli, but also decreased exosome release in podocytes of WT/WT mice and Smpd1trg/Podocre mice. However, GW and Rap only blocked exosome‐mediated IL‐1β secretion from podocytes, but had no effect on inflammasome formation or activation in glomeruli during hHcy. However, all 3 treatments attenuated the inflammatory response in glomeruli and associated injury as shown by reduced immune cell aggregation, decreased proteinuria and ameliorated glomerular sclerosis. These results suggest that without enhanced exosome release NLRP3 inflammasome activation in podocytes may not produce local glomerular inflammatory response and associated injury during hHcy.

Support or Funding Information

This study was supported by NIH grants DK54927 and DK102539.
Assessing Bioavailability of Alaskan Blueberry Botanicals in vitro
Authors: Owen Savanah Renee , Collin Aline , Kuhn Thomas
Published: April 2020

Prevalence of type II diabetes, a loss of insulin sensitivity, is rapidly increasing in rural Alaskan communities likely due to increasing obesity rates. However, there is a lower percentage of cases when compared to the incidence of type II diabetes in the contiguous US. This phenomenon could possibly be a consequence of berry‐rich diets as part of the traditional subsistence lifestyle of rural Alaskan populations. Wild Alaska blueberries, specifically, have been shown to improve or rescue insulin sensitivity in vitro. However, it is imperative to understand bioavailability, efficient uptake from the intestinal lumen into the bloodstream, to correlate these in vitro findings to in vivo situations. For this project, we modified an in vitro Caco‐2 bioavailability assay in order to mimic botanical transport by the intestinal lining. Caco‐2 monolayers used in these assays were tested for accuracy and intactness using both microscopy and transport studies. Transport studies were used to ensure monolayer integrity both prior to and after botanical transport, using 3kDa fluorescent dextran and Lucifer yellow as the control reporter molecule respectively. Results from this study provided insight into cell culture and cytotoxicity of botanical extracts, which can be used to improve upon the botanical bioavailability assay. Transported botanical extract from successful transport studies will be analyzed via LC‐MS and compared to a full, untransported extract control.

Support or Funding Information

Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Numbers UL1GM118991, TL4GM118992, or RL5GM118990. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103395. The content is solely the responsibility of the authors and does not necessarily reflect theofficial views of the NIH.
Genetic Testing of a Puerto Rican Family Trio with Oculocutaneous Albinism Type 1B Reveals a Misdiagnosis of Hermansky‐Pudlak Syndrome
Authors: Serrano Joseline , Montes Ingrid M. , Renta Jessicca Y. , Rojas Ricardo , Cadilla Carmen L.
Published: April 2020

Hermansky‐Pudlak syndrome (HPS) is a rare autosomal recessive disorder where the biogenesis of lysosomal‐related organelles is affected. Clinical manifestations depend on the type of HPS, and frequently include a hemorrhagic diathesis, oculocutaneous albinism and a lysosomal storage disease. Some types of HPS manifest with pulmonary fibrosis, granulomatous colitis, neutropenia, immunodeficiency and neurological symptoms. Up to date, 10 types of HPS have been reported, each arising from mutations in different genes. In Puerto Rico, HPS is caused primarily by founder mutations in the HPS1 and HPS3 genes. The present study aims to determine the genetic defects in a Puerto Rican family trio that does not harbor the HPS1 and HPS3 founder mutations. The proband presented with ocular albinism, reduced number on platelet dense bodies, neutropenia, and gastroesophageal reflux disease, which led clinicians to diagnose her with HPS. Exome sequencing was performed for the family trio using blood samples, where single nucleotide variants in several HPS genes as well as the Tyrosinase gene (TYR) were found. The proband carries three TYR gene amino acid changes: G47D, S192Y, and R402Q. The pathological G47D mutation has been previously detected in Puerto Rican oculocutaneous albinism type 1 (OCA1) patients who were homozygous for these mutations. The proband’s father carries the G47D and S192Y variants, but the R402Q is a de novo mutation in the proband. The R402Q amino acid change is frequently found in Caucasians (MAF=.252) and Puerto Ricans (MAF=0.183) and makes TYR a temperature‐sensitive enzyme. The TYR gene mutations detected would imply that the proband has very low TYR activity. Both the S192Y and the R402Q variants have been proposed to have combinatory variant effects with other more severe OCA1 mutations. Our findings suggest that the combination of common and pathological TYR alleles may be the cause of the proband’s albinism, and that the other clinical manifestations may be unrelated or due to the other mutations found in HPS genes. This study supports previous work that proposed that the common S192Y and R402Q TYR alleles combined with a deleterious mutation can cause OCA1B, but their clinical relevance still need to be investigated further since there is also evidence that homozygosity for R402Q does not cause OCA. The clinical relevance of supposedly benign HPS gene SNPs also deserves further studies.

Support or Funding Information

This research was supported in part by RCMI grants U54 MD007600 and U54MD007587 (National Institute on Minority Health and Health Disparities), RISE grant R25GM061838 and PR INBRE grant P20GM103475 (National Institute of General Medical Sciences).
Essential Anatomy for Anesthesiology in the Undergraduate Medical Curriculum
Authors: Harmon Derek , Hankin Mark , Martindale Jim , Davies David , McNary Thomas , Sanky Charles , Laitman Jeffrey , Reidenberg Joy S , Keim Sarah , Miriyala Sumitra , Pratt Rebecca , Lopez Lisa , Doroudi Majid , Topping Daniel , Royer Danielle , Cotter Meghan
Published: April 2020

Introduction

To prepare medical students for clinical training and practice, it is critical to understand the anatomical knowledge considered most important for different clinical specialties.

Aim

To address this issue, a consortium of anatomists in the US and Canada is collecting data from clinical educators in Anesthesiology clerkships and electives to identify the anatomy they consider essential.

Methods

An IRB‐approved, online survey (Qualtrics, Seattle, WA) was used to assess the importance of 98 anatomical topics in seven body regions. The study first examined the percentage of Anesthesiology clinical educators (clerkship/elective directors and attending physicians) that considered each anatomical region important to their specialty. Second, the study examined the rank assigned to each anatomical topic using an ordinal scale from 1 (not important) to 4 (essential).

Results

At the time of abstract submission, data had been collected from 21 Anesthesiology clinical educators at 14 medical schools. The percentage of Anesthesiology clinical educators that considered each anatomical region important to their specialty were (highest‐to‐lowest): Back (100%), Head & Neck (100%), Thorax (95%), Upper Limb (86%), Lower Limb (86%), Abdomen (80%), and Pelvis & Perineum (50%). Further data analysis has identified the highest ranked anatomical topics in each body region for the Anesthesiology clerkship/elective.

Discussion and Conclusion

This database provides detailed information regarding the most clinically relevant anatomical topics as identified by Anesthesiology clinical educators. This information can aid in focusing preclinical learning to best prepare medical students for success in their undergraduate and graduate clinical experiences.
Characterization of Sodium Bicarbonate Transporters NBCe1 and NBCn1 as CO2 channels
Authors: Wang Dengke , Boron Walter F.
Published: April 2020

The known “gas channels” are members of the AQP and Rh families, and appear to conduct CO2 mainly through the stable hydrophobic central pores of these multimeric proteins. The present work helps to define a new class of gas‐conduction pathway: transporters that may conduct CO2 through cracks that form transiently as the protein cycles through various conformations—much like H2O can leak through certain membrane proteins. To identify CO2 channels, we developed a novel tool—expressing AQP5 in Xenopus oocytes, followed by injection of small amounts of carbonic anhydrase II (CAII), and then exposure of the cell to CO2/HCO3− solutions. Cytosolic CAII promotes the consumption of incoming CO2, thereby maintaining a low [CO2] at the inner surface of the cell membrane, and maximizing the gradient for CO2 influx. A semiquantitative measure of this CO2 influx is the spiking increase in surface pH (DpHS) that occurs as CO2 enters the cell, lowers [CO2] at the outer surface of the cell membrane, causes some HCO3− in the vicinity of the membrane to undergo the reaction HCO3− + H+ ® H2O + CO2, and thereby replenishes some of the lost CO2. The pHS spike is greatest near time‐zero, when the inward CO2 gradient—and thus the reaction HCO3− + H+ ® H2O + CO2—is greatest, and then exponentially wanes as CO2 equilibrates across the cell membrane. Oocytes expressing AQP5 and then injected with Tris buffer (but not CAII) have a much greater DpHS than controls injected with H2O (not cRNA) and Tris (not CAII), confirming that AQP5 is an excellent CO2 channel. Injecting a small amount of CAII (dissolved in Tris) into oocytes not expressing AQP5 produces only a slight increase in DpHS because the rate‐limiting step is membrane CO2 permeability. However, for AQP5‐expressing oocytes, injecting this same small amount of CAII produces a very large increase in DpHS. In other words, the CAII‐dependent change in spike height—D(DpHS)—is much greater in AQP5‐expressing oocytes than H2O‐injected controls. We conclude that the measured D(DpHS) in ±CAII experiments is a powerful tool for assessing CO2 conductance. We applied this method to measuring the CO2 conductance of Na/HCO3 co‐transporters, NBCe1‐A and NBCn1‐G. Preliminary data show that, in the presence of cytosol CAII, D(DpHS) is >1.5‐fold greater in the oocytes expressing NBCe1‐A than the H2O‐injected controls, supporting the hypothesis that NBCe1‐A has a CO2 conductance. Similarly, D(DpHS) is >5‐fold greater in the oocytes expressing NBCn1‐G than the H2O‐injected controls, supporting the hypothesis that NBCn1‐G has a CO2 conductance.

Support or Funding Information

Supported by grants GM111251 (the U grant) and N000141612535 (the MURI grant)
Estimates of Motor Neuron Excitability During Postural Sways in Lower Leg Muscles
Authors: Khurram Obaid U. , Negro Francesco , Heckman Charles J. , Thompson Christopher K.
Published: April 2020

Motor system investigations are often performed under isometric conditions, but real‐world motor behaviors are seldom isometric. During tasks where isometric force or torque measurements are not feasible (i.e. during unconstrainedconditions), EMG is often used as a surrogate of the level of muscle activity. However, the linear relationship between EMG and force generation breaks down during non‐isometric contractions. It is not clear how this affects investigators’ ability to discriminate motor units using state‐of‐the‐art high‐density surface EMG arrays. Additionally, the paired motor unit analysis technique, which generates an estimate of the intrinsic excitability of a ‘test’ motor unit by determining discharge rate hysteresis (ΔF), has not been evaluated in unconstrained conditions. Consequently, we asked if 1) visual feedback provided by the subject’s own EMG activity allow for subjects to adequately perform triangular ramp contractions in constrained conditions and 2) if the EMG visual feedback is provided in unconstrained conditions, are ΔF values in subjects similar to those in the constrained condition? We hypothesized that during voluntary isometric ramp contractions, ΔF will be similar regardless of the feedback source. We further hypothesized that standing postural adjustments using EMG feedback in unconstrained conditions will produce similar ΔF values to those calculated during the isometric ramps. We expected that although the mean values across conditions will be similar, the inter‐subject coefficient of variation of ΔF will be greater in the less constrained condition. Accordingly, surface EMG using high‐density array electrodes was collected from five subjects under three conditions: 1) seated in a dynamometer in the isometric mode with torque feedback, 2) in the isometric dynamometer with filtered EMG feedback, and 3) during postural adjustments while standing freely with EMG feedback. In agreement with our hypothesis, we found that ΔF values across all three conditions were similar. In the constrained conditions with either torque or EMG feedback ΔF was 3.10 ± 0.70 imp/s and 3.40 ± 0.75 imp/s, respectively; during the unconstrained case with EMG feedback, ΔF was 3.08 ± 0.73 imp/s (all n = 15, mean ± SE). Contrary to our expectations, the coefficient of variation was not greater in the unconstrained standing condition. These results demonstrate that real‐time EMG feedback is sufficiently similar to torque feedback during both isometric and near‐isometric, unconstrained contractions, suggesting that detailed investigations of motor unit activity may be expanded to more functional movements and used relatively easily in more clinical settings.
Chronic Stress Alters Expression of Vomiting‐related Receptors in CNS in SD Rats
Authors: Li Zhuman , Xu Feng
Published: April 2020

Chemotherapy‐induced nausea and vomiting (CINV) are extremely painful side effects that occur frequently in cancer patients during chemotherapy. Our previous study found that cancer patients have high incidence of depression that correlate positively with CINV. However, whether depression aggravate the CINV is not clear up to now. In this report, we established chronic unpredictable mild stress‐induced depression model in SD male rats. The vomiting‐related receptors including serotonin 3 receptor (5‐HT3R), dopamine receptor (DA2R), endogenous cannabinoid receptor (CB1R) and substance P receptor (NK1R) in the posterior region of the fourth ventricle and in the medulla were studied by qRT‐PCT and Western blotting analysis. Blood levels of 5‐HT, DA, AEA and substance P were measured by ELISA. There were significant difference in behavioral evaluation scores between the model rats and control rats, indicating depression model was successfully built up in model rats. Compared with control rats, blood levels of 5‐HT, DA and substance P were significantly decreased while AEA was significantly increased in model rats, the mRNA and protein of expression levels of 5‐HT3R, DA2R, NK1R were increased while CB1R were decreased in model rats. In conclusion, chronic stress altered expression of vomiting‐related receptors in CNS in SD rats.

Support or Funding Information

Shanghai Science and Technology Commision Plan Project (No. 19411971700)
Mechano‐Electric Coupling and Arrhythmogenic Current Generation in a Multi‐Scale Computational Model of Coupled Myocytes
Authors: Timmerman Viviane
Published: April 2020

Heterogeneous mechanical activity during acute myocardial ischemia is thought to contribute to arrhythmogenic alterations in cardiac electrophysiology. Stretch‐induced perturbations to cardiomyocyte electrophysiology may trigger arrhythmias via a variety of mechano‐electric coupling (MEC) mechanisms. While the role of stretch‐activated ionic currents has been investigated intensively using computational models, experimental studies have shown that mechanical strain can also trigger intra‐ and inter‐cellular calcium waves. To assess whether the inherent stretch dependence of myofilament calcium affinity may promote ischemia‐induced arrhythmogenic intra‐ and inter‐cellular calcium waves, we coupled a mathematical model of excitation‐contraction coupling in rabbit ventricular myocytes to a model of myofilament activation and force development. The model was modified to mimic observed electrophysiological alterations during ischemia, and was subjected to systolic stretch.

We compared a zero‐dimensional (0D) and one‐dimensional (1D) compartmental analysis, for which multiple myocytes were electrically and mechanically coupled in series. In the 1D model each myocyte consisted of 50 bi‐directionally coupled electromechanical models in addition to calcium diffusion and strain transfer between adjacent units. Both, the 0D and 1D model, were fitted to calcium alterations during homogeneous stretch transient. The addition of end‐to‐end mechanical interactions of inter‐ and/or intracellular sarcomeres enabled the model to capture: (1) the effects of MEC on intracellular calcium dynamics at the sarcomeric scale; and (2) the effects of mechanical heterogeneities on calcium dynamics at the cellular scale.

The simulation results suggest that in the 0D only the triggers of calcium‐mediated delayed after depolarizations (DADs) could be observed. In the 1D model, in conditions of calcium overload and only for the non‐ischemic myocytes, an increase in stretch or an increase in sarcomere heterogeneity increase the susceptibility for myofilament triggered calcium waves causing DADs. Additionally, the specific sarcomere heterogeneity can modulate intracellular calcium dynamics which may increase susceptibility to pro‐arrhythmic intra‐ and inter‐cellular calcium wave propagation, but only for spatially explicit myocyte models.
Codeine Reconfigures The Respiratory Network In The Cat
Authors: Bolser Donald C. , Segers Lauren S. , Nuding Sarah C. , Baekey David M. , Morris Kendall F.
Published: April 2020

The mechanisms responsible for depression of breathing by opioids are poorly understood. Multiple regions of the brainstem have been implicated in respiratory depression, including the pre‐Bötzinger complex and pons. At the network scale, evidence exists that opioids act at sites that are presynaptic to medullary premotor and motoneurons. We speculated that opioids reconfigure the respiratory network at doses that are subthreshold for depression of breathing. Cats (n=11) were decerebrated, paralyzed, and artificially ventilated. Extracellular action potentials of neurons in the region of the nucleus of the tractus solitarius (NTS), raphe and ventral respiratory column (VRC) were recorded in the brainstem from multiple electrode arrays. Codeine (0.009–4.9 mg/kg) was administered via the vertebral artery. The discharge identity (pattern of discharge during breathing) of 63/148 recorded neurons changed after codeine administration relative to control. Of the 56/168 neurons that were not breathing modulated, 31/56 (55%) became respiratory modulated after codeine. Ninety‐two neurons were respiratory modulated in the control period and 32 (35%) of these either were not modulated during breathing or altered their respiratory pattern after administration of codeine. Cross correlation analysis indicated that functional correlations consisting of offset peaks/troughs or central peaks were detected during the control period between 56 pairs of neurons whereas 75 pairs were correlated after codeine. Feature maps of putative circuits in each experiment suggested significant reorganization of the respiratory network after codeine. Clusters of VRC inspiratory neurons exhibited significant alterations in functional correlations consistent with altered synchrony. Features in cross correlation histograms during codeine administration typically appeared early in the dose response rather than being restricted to the highest dose, consistent with a high sensitivity to the drug. Most of the features in cross correlation histograms appeared at doses that were lower than those that produced significant depression of breathing. These results support opioid‐induced reconfiguration of the respiratory network. Alternate hypotheses regarding the functional manifestation of this reconfiguration include a) compensatory responses of the network as sensitive neuron populations are affected by increasing doses of opioids, and b) dysregulation of synchrony as a prerequisite to respiratory depression.

Support or Funding Information

Supported by HL131716