bluuebunny/crossref_metadata_2025 huggingface dataset:
Histology Education in an Integrated, Time‐restricted Medical Curriculum – Academic Outcomes and Students’ Study Adaptations
Authors: Hortsch Michael , Gribbin William , Wilson Eric , McTaggart Suzy
Published: April 2020

In an ever‐changing medical curricular environment, time dedicated for anatomical education has become more and more curtailed. This happened at the University of Michigan Medical School when the period of preclinical medical education was reduced from two years to one year starting with the academic year 2016–17. Histology instruction remained integrated in organ system courses, but all faculty‐guided laboratory sessions were completely removed, reducing histology teaching to a lecture‐only format and requiring students to study virtual histological slides from a course website on their own time. In accordance with the shortened instructional time, the number of histology examination questions was reduced more than twofold. We investigated changes in students’ histology examination results, surveyed their motivation to learn histology, and assessed their use of histology educational material and opportunities before and after these curricular changes were implemented. Students’ motivation to learn histology and their evaluation of histology lectures slightly increased in the new curriculum. However, they devoted less study time to learning histology and used the course website and supplementary learning material less than students in the old curriculum. Students’ cumulative histology examination scores over the entire M1 year were significantly lower in the new curriculum and the number of students with overall scores under 75%, which is considered a failing performance, increased more than fifteenfold. Aside from the shorter instruction time, the decision to not provide histology performance subscores to students may also have contributed to this decrease in academic performance. In addition, we suspect that a decentralization of curricular organization, leaving decisions to individual organ system course directors about the integration and time dedicated for longitudinal histology instruction, may have played a role in decreasing consistent histology education in the new curriculum. This study received a non‐regulated status from the University of Michigan IRB (HUM00162947).
Capillary Oxygen Triggers Conducted Hyperpolarization and Dilation Independent of Extracellular K+ and Endothelial KIR2.1
Authors: Kowalewska Paulina M. , Milkovich Stephanie , Ellis Christopher G. , Welsh Donald G.
Published: April 2020

The matching of red blood cell (RBC) supply to oxygen demand is an intricate process which requires generation of a capillary stimulus and triggering of a transduction process that dilates upstream arterioles. The sensor’s identity, the stimulus it generates and the mechanisms that enables it are openly debated and a source of active inquiry. This study tested whether extracellular K+ is the stimulus and if capillary KIR2.1 channels enable upstream arteriolar dilation via conduction. Experiments were performed in vivo, using a live microcirculatory preparation (extensor digitorum longus); the local oxygen environment was tightly controlled (custom stage insert) and capillary RBC kinetics monitored in mice lacking endothelial KIR2.1 or connexin 40. Dropping PO2 on the muscle surface (53 mmHg to 15 mmHg or 0 mmHg (3 min)) induced a stark microvascular response in control animals (RBC supply rate, velocity and hematocrit increased by 56%, 28% an 18%, respectively) without diminishing mitochondrial respiration. This blood flow response failed to materialize in connexin 40−/− mice. As this response was absent, capillary RBC O2 saturation was lower under normal conditions in the connexin 40−/− animals. In contrast, endothelial KIR2.1−/− mice reacted normally to O2 challenges, with RBC supply rate, velocity and hematocrit rising by 43%, 28%, and 11%, respectively. These findings show that coupling O2 demand‐to‐O2 supply requires coordinated electrical signaling among cells connected by gap junctions comprised of connexin 40. They also show that endothelial KIR2.1 channels are not responsible for initiating the hyperpolarization needed to drive conduction. These findings begin the process of restructuring our understanding of blood flow regulation and how O2 initiates this process independent of metabolite production.

Support or Funding Information

This work was funded by NSERC Discovery grants to CGE and to DGW.
Vascular Dysfunction and Posttraumatic Stress Disorder: Examining the Role of Oxidative Stress and Sympathetic Activity
Authors: Weggen Jennifer B. , Autler Aaron S. , Hogwood Austin C. , Darling Ashley M. , Decker Kevin P. , Garten Ryan S.
Published: April 2020

Purpose

The physiological manifestations of posttraumatic stress disorder (PTSD) have been associated with an increase in risk of cardiovascular disease (CVD) independent of negative lifestyle factors. The goal of the study was to better elucidate the mechanisms behind the increased CVD risk by examining peripheral vascular function, a precursor to CVD. Moreover, this study sought to determine the role of oxidative stress and sympathetic nervous system (SNS) activity in PTSD‐induced vascular dysfunction.

Methods

Sixteen individuals with PTSD (10 women, 6 men; age 24 ± 4 years), and twenty‐four healthy controls (CTRL; 15 women, 9 men, 24 ± 4 years), participated in the study. The PTSD group participated in two visits, consuming either a placebo or antioxidant cocktail (AO ‐ vitamins C and E and alpha lipoic acid) prior to their visits, in a randomized order. Arm vascular function was assessed via the reactive hyperemia‐induced flow mediated dilation of the brachial artery (BAFMD) technique and evaluated with Doppler ultrasonography. Brachial artery and arm microvascular function were determined by percent change of diameter from baseline normalized for BA shear rate (BAD/Shear), and blood flow area under the curve (BF AUC), respectively. Heart rate variability (HRV) was used to assess autonomic nervous system activity.

Results

BF AUC was significantly lower (p = 0.02) and SNS activity was significantly higher (p = 0.003) in the PTSD group when compared to the CTRL group. BAD/Shear was not different between groups. Following the acute AO supplementation, BF AUC was augmented and SNS activity was reduced, to which it was no longer significantly different (BF AUC: p = 0.16, SNS activity: p=.41) when compared to the CTRL group.

Conclusion

Young individuals with PTSD demonstrated lower arm microvascular, but not brachial artery, function as well as higher sympathetic activity when compared to healthy controls matched for age, sex, and physical activity level. Furthermore, this microvascular dysfunction and SNS activity was attenuated by an acute AO supplementation to the level of the healthy controls. Taken together, this study revealed that the modulation of oxidative stress, via an acute AO supplementation, improved vascular dysfunction in individuals with PTSD, potentially by reducing the substantial SNS activity associated with this disorder.
Characterizing the Effect of the Hypoxic Vasoconstriction Response Curve on Blood Flow Distribution and Oxygenation in the Lung
Authors: Roy Tuhin K. , Secomb Timothy W.
Published: April 2020

The ability of the lung to take up oxygen can be characterized by its effective diffusing capacity, which is determined by the transport characteristics of the alveolar‐capillary membrane as well as heterogeneity of pulmonary blood flow. At rest, the healthy lung has a high degree of tolerance for pulmonary flow heterogeneity, but under conditions of physiological stress such as exercise, oxygen uptake may be inadequate to satisfy tissue demand if flow is very heterogeneous. We previously used a mathematical model of pulmonary oxygen uptake to examine the ability of hypoxic pulmonary vasoconstriction (HPV) to mitigate flow heterogeneity. In the model, flow heterogeneity is characterized by the coefficient of variation (CV) of pulmonary capillary blood flow. Assuming an initial lognormal distribution of blood flow with a normal level of heterogeneity (CV = 1.66), we simulate flow regulation over a range of oxygen consumption from rest to maximal exercise by adjusting pulmonary flow resistance in each vessel in accordance with an HPV response curve obtained from previously published experimental data. The effect of HPV on vessel flow resistance is assumed to be characterized by a sigmoidal response dependent on the capillary oxygen tension, which is approximated as the mean of the venous and arterial values in each pulmonary capillary; its maximal sensitivity occurs at a stimulus oxygen tension of approximately 30 torr. The purpose of the present study was to determine whether altering the shape of the HPV response curve would result in an improvement in CV reduction. Altering the HPV by shifting the sigmoidal curve to the right by 10 or 20 torr worsened CV, extraction and total pulmonary vascular resistance (PVR). Similar detrimental results were obtained when the range of oxygen tensions over which HPV varied was expanded by a factor of two, increasing the oxygen tension at which the sensitivity of HPV to oxygen tension was maximal. On the other hand, shrinking the range of variation of the HPV curve by a factor of two neither improved nor significantly worsened the CV or extraction, and resulted in a PVR distribution similar to the case of the normal HPV curve. These results demonstrate that the observed shape of the HPV response tends to minimize heterogeneity of pulmonary flow under conditions of exercise.

Support or Funding Information

Supported by NIH U01 HL133362.
A Single Heat Exposure Improves Heat Tolerance in a Mouse Exertional Heat Stroke Model
Authors: Ward Jermaine Allen , Dineen Shauna , Plamper Mark , Mayer Thomas , Leon Lisa
Published: April 2020

Exertional heat stroke (EHS) is an ongoing medical issue for military personnel. Currently there is poor insight into the etiology of organ damage sustained during EHS and what factors may increase susceptibility. Recent evidence suggests that prior heat injury can increase the likelihood of subsequent EHS. Individuals that have experienced prior EHS report being less tolerant to heat and are flagged in the military for extra surveillance when conducting training exercises. The goal of this study was to determine the impact of prior EHS on heat tolerance and running performance during a subsequent EHS event. Using C57BL/6J male mice, we hypothesized that prior EHS would decrease thermoregulatory performance in the heat, as measured by total heating time to collapse (min), total distance run (m), maximal speed achieved (m/min), and thermal load (°C·min) compared to those without prior EHS. Using a forced running EHS protocol (environmental temperature [Tenv]=37.5 ± 0.2°C), mice were allowed to recover for 24 hours (EHS 24h; n=31), 3 days (EHS 3D; n=22) or 7 days (EHS 7D; n=21) before being subjected to a secondary EHS. To our surprise, mice exposed to two EHS events exhibited a significant increase in time to collapse, distance ran, and thermal load compared to the single EHS group. Mice in the EHS 24h group performed best in three of four performance measures; heating time to collapse (344.55±8.89 min), distance ran (1908.21±92.30 m) and max speed (8.71±0.23m/min) during their second EHS. EHS 24h mice also displayed the highest maximum core temperature (42.50 ± 0.3°C, Tc Max) prior to loss of consciousness, which was significantly higher than the other groups (p<0.02). EHS 3D mice had the highest thermal load (1035±30.8°C·min) compared with the EHS 7D group only (930.75±24.65°C·min, p<0.01). Overall the EHS 24h mice had the best performance and showed the highest increase in performance from their initial EHS, suggesting an increase in heat tolerance. The EHS 3D and EHS 7D group performance measures showed a decay over time, regressing towards initial heat performance levels. Author views not official US Army or DoD policy.
Mechanosensitive Mechanism of Lung Regeneration through Endothelial YAP1
Authors: Hendee Kathryn , Mammoto Tadanori , Mammoto Akiko
Published: April 2020

Angiogenesis, the formation of new blood capillaries, plays a key role in organ development, homeostasis and regeneration. In addition to chemical factors, mechanical forces control angiogenesis and organ regeneration. We have reported that the mechanosensitive transcriptional co‐activator Yes‐associated protein (YAP1) in endothelial cells (ECs) is necessary for post‐pneumonectomy (PNX) lung growth. Right pulmonary artery (PA) pressure and shear stress increase after left unilateral PNX. However, it remains unknown whether changes in PA pressure and shear stress after PNX control post‐PNX regenerative lung growth through endothelial YAP1 signaling. Here we found that ligation of the right cardiac lobe PA after left PNX further increases right PA pressure and shear stress, upregulates expression of YAP1 and angiogenic factor Tie2, and stimulates post‐PNX lung growth in the remaining non‐occluded lobes. These effects are attenuated in VE‐cadherin‐specific Yap1 knockout mice. Hydrostatic pressure or shear stress applied to cultured human pulmonary arterial ECs also increases the expression and activity of YAP1 and induces EC proliferation and migration in vitro. These results suggest that increases in the right PA pressure together with elevation in shear stress after left PNX stimulate angiogenesis and post‐PNX lung growth through endothelial YAP1. Modulation of the mechanical environment or endothelial YAP1 expression/activity could be an efficient strategy for regenerative lung growth.

Support or Funding Information

NIH RO1HL142578
Optimal Cutting Temperature Medium‐Embedding Is a Valid Method for Storing and Preparing Myocardial Biopsies Preceding Myofilament Function‐Assessment
Authors: Isackson Henrik , Ng Henry , Agic Mediha Becirovic , Hultström Michael
Published: April 2020

Background and objective

Optimal Cutting Temperature (OCT) media is commonly used for histological characterisation. Myocardial tissues from clinical biopsies are precious and thus utilising them for both histological and functional assessment is an attractive prospect. This study was designed to assess whether OCT media stored samples can be used for preparing myocardial tissue for myofilament function‐assessment.

Methods

Liquid nitrogen (LN) – and OCT‐stored myocardium from mouse was compared using a permeabilised fibre set‐up for functional characterisation of thin and thick filament‐interaction including Ca2+ sensitivity (pCa50), cooperativity along the thin filament (nH), cross‐bridge cycling rate (ktr), Ca2+ activated maximum tension (Fmax)/cross sectional area, cross‐bridge recruitment upon Ca2+ activation, as well as passive strain (kstrain) and the relative titin contribution to passive stiffness. LN samples were prepared in a conventional fashion and OCT‐stored tissue was first sectioned in parallel with papillary muscle longitudinal axis before chemical demembranisation and further trimming under the microscope.

Results

Ca2+ sensitivity (pCa50): LN (n=8): 5.75 ±0.019, OCT (n=8): 5.69 ± 0.022 SEM (p>0.05). Cooperativity along the thin filament (nH): LN (n=8): 2.63 ± 0.24, OCT (n=8): 2.53 ± 0.25 SEM (p>0.05). Fmax: LN (n=8): 19.6 ± 1.9, OCT (n=8): 11.6 ± 1.7 mN/mm2 SEM (p<0.05). Ktr: LN (n=8): 37.5±1.6, OCT (n=8): 37.9 ± 1.6 s−1 SEM (p<0.05). Relative cross‐bridge recruitment upon full Ca2+ activation: LN (n=8): 0.32 ± 0.015, OCT (n=7): 0.31 ± 0.014 SEM (p<0.05). kstrain: LN (n=7): 13.9 ± 0.67, OCT (n=7): 11.4 ± 0.49 L−1, p<0.01). Relative titin contribution to stiffness at 20 % stretch from slack: LN (n=6): 0.52 ± 0.07, OCT (n=7): 0.80 ± 0.03 SEM, (p<0.01).

Conclusions

OCT‐storage and cryostat sectioning before mounting fibre bundles on a force‐measuring rig can be used for investigating relative measures of function depending on active force development, ie pCa‐tension relationship, cooperativity along the thin filament and cross‐bridge cycling rate. There appears to be no negative effect on the amount of cross‐bridge recruitment upon Ca2+ activation compared to a conventionally LN‐stored and prepared sample. Absolute values such as Fmax, as well as passive strain, need to be interpreted with great caution as they clearly differ between mode of preparation and thus these parameters are not suited for investigation. We speculate this may be due to how cutting of fibre bundles using a cryostat, to an unknown degree, disrespects the natural continuity of the bundles and thus hampers force relay.

Support or Funding Information

This work was supported by: Swedish Stately Grant for Medical Research (ALF‐means) & Lars Hierta Memorial Fund
High Intensity Intervals Expand Plasma and Improves Exercise Performance in Acute Hypoxia
Authors: Gillum Trevor , Gorini Felipe
Published: April 2020

Introduction

Hypoxia causes decrements in submaximal and maximal exercise performance. Hypervolemia may improve O2 delivery to the metabolically active tissue, thus lowering physiological and cardiovascular strain during moderate exercise at altitude. Since significant time is needed to acclimate to exercise in hypoxia, we sought to expand plasma volume (PVE) through a single bout of high intensity intervals (HII) in an attempt to expedite improvements in aerobic performance.

Purpose

To determine if the hypervolemic response to a single bout of HII was sufficient to mitigate the hypoxia associated declines in exercise capacity.

Methods

In our randomized counterbalanced study, 7 males (24.4 ± 5.8 years, VO2max 46.9 ± 5.8 ml kg−1 min−1, 12.8 ± 4.4% body fat) performed two 15km cycling time‐trials (TTs) in normobaric hypoxia (FiO2 = 15%) before and 48 hours following HII (8x4 minutes at 85% VO2max with 4 minutes rest between rounds) or a control bout (CON) performed at 50% VO2max, in which duration was extended to assure identical kilojoules completed between conditions. Pre‐exercise blood samples were collected to quantify changes in plasma volume, while hemodynamic data were collected utilizing PhysioFlow.

Results

Increased PVE was observed 24 hours (6.96% ± 4.84%) and 48 hours (9.77% ± 4.26%) (p<0.05) following HII while the CON condition decreased plasma volume 48 hours post (−3.75% ± 2.62%) (p<0.05). Under the HII condition, participants showed an improvement in TT performance (Time: 1880 ± 215s to 1840 ± 203s, Power: 164.8 ± 41.2W to 171 ± 39.5W) (p < 0.05) and lowered HR (164.5 ± 9.5 bpm to 161.9 ± 8.8 bpms) (p < 0.05). Stroke volume (SV) and cardiac output (Q) manifested an upwards trend during TT performance within the HI condition (p = 0.09 and p = 0.08, respectively). There was no difference in performance, Q, or SV in the CON condition. Hydration, RPE, SaO2, and blood lactate were similar in both TTs in hypoxia.

Conclusion

A single bout of HII resulted in increased cycling performance in acute hypoxia, accompanied by an enhanced PVE both 24 and 48 hours following exercise. This observed enhancement in cardiac efficiency following intense exercise may be desirable in military populations or individual needing to complete physical tasks at moderate altitude without the means or time to fully acclimatize.
ANNONA MURICATA NON‐SELECTIVELY SUPPRESS PC3 CELLS by INHIBITNG CELL PROLIFERATION and INDUCING CELL CYCLE ARREST
Authors: Vora Akshay P. , Gugnani Kuljeet S. , Rondón-Ortiz Alejandro Nico , Pino-Figueroa Alejandro
Published: April 2020

Annona muricata (AM) also known as graviola or soursop is a tropical evergreen tree that belongs to family Annonaceae. The leaves of the tree have various therapeutic benefits against numerous diseases including various multi drug resistant cancers. The major phytochemical constituents associated with its anticancer activity are annonaceous acetogenis (AAGs). It was hypothesized that AAGs exhibit their anticancer activity by possibly inhibiting mitochondrial NADH‐ubiquinone complex which limits the ATP supply to the cancer cells. However, the exact mechanism is unknown. The purpose of the study was to determine whether ethyl acetate fraction (EAF) is selectively kills prostate cancer cells and the mechanism by which the EAF induces cell death or reduces cell proliferation.3‐(4,5‐dimethylthiazol‐2‐y1)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfopheny1)‐2H‐tetrazolium (MTS) cell viability assay was performed on both cancer and healthy cells and it was observed that EAF non‐selectively reduces cell viability of PC3 cells and non‐cancer cells. To further examine the effect of EAF on PC3 cells, cell proliferation assay was performed, and it was observed that EAF inhibits cell proliferation in PC3 cells. Western blot analysis for cyclin D1 expression level was performed and EAF exposed cells showed reduced cyclin D1 levels suggesting that AM induces cell cycle arrest in PC3 cells. Immunoblotting analysis suggested that there was no significant effect on poly (ADP ribose) polymerase 1 (PARP‐1) expression levels however EAF significantly increases c‐PARP expression levels indicating that EAF does not induce apoptosis in prostate cancer cells. These results suggest that EAF reduces cell viability of PC3 cells by inhibiting cell proliferation via inducing cell cycle arrest.
Disruption of estrogen regulation of orexin neurons: a novel mechanism in the pathophysiology of respiratory manifestations of panic disorder in females
Authors: Tenorio-Lopes Luana , Fournier Stéphanie , Bretzner Frédéric , Kinkead Richard
Published: April 2020

Panic disorder (PD) is an anxiety disorder that affects ~5% of the population. A significant group of PD patients are characterised by excessive behavioral and physiological responses to CO2 inhalation. The prevalence of PD is 2 to 3 times more frequent in women yet, the origins of this sexual dimorphism are unknown; the fact that excessive responsiveness to CO2 inhalation peaks during the pre‐menstrual phase suggest a role of ovarian hormones. Early life stress is a significant risk factor for PD and there is growing evidence indicating that stress‐related increase in the function of orexin‐producing neurons (ORX) is key to PD. Based on data suggesting that 17b‐estradiol (E2) inhibits ORX, we tested the hypothesis that insufficient E2‐regulation of ORX neurons contributes to stress‐related respiratory manifestations of PD in females. Experiments were performed on adult female rats previously subjected to neonatal maternal separation (NMS; 3h/day postnatal days 3 to 12) or raised under standard conditions. In this model, NMS females show an excessive hyperventilatory response to CO2 by comparison with controls; this effect is greatest during the proestrus phase. Inactivation of ORX1 receptors by SB 33486 (15 mg/kg; ip) prevents this effect. Whole cell recordings (voltage clamp) were performed on GFP‐labeled ORX neurons by AAV microinjection. Excitatory post‐synaptic currents (EPSCs) converging onto ORX neurons were recorded under basal conditions and following E2 bath application (100nM; 10 min). Monitoring of the estrus cycle showed that in control females, EPSC frequency was inversely proportional to circulating E2 levels associated with each phase. This relationship was not observed in NMS females; EPSC frequency of NMS females was greater than controls during the proestrus phase. E2 application reduced EPSC frequency (but not amplitude); however, this effect was not affected by stress or the estrus cycle phase. We conclude that NMS disrupts E2‐mediated inhibition of ORX neurons. This is a novel mechanism in the pathophysiology of PD.

Support or Funding Information

Supported by an operating grant from the Canadian Institutes of Health Research
Physiological Adaptations During the Acclimatization To‐ and Deacclimatization From‐Chronic Hypercapnia
Authors: Buchholz Kirstyn , Burgraff Nicholas , Neumueller Suzanne , Pan Lawrence , Hodges Matthew , Forster Hubert
Published: April 2020

Respiratory disturbances, such as chronic obstructive pulmonary disease (COPD), are often associated with chronically elevated arterial CO2 (PaCO2 >45mmHg), which has been linked to an increase risk of all‐cause mortality. Additionally, acute exacerbations of respiratory symptoms may lead to greater levels of PaCO2 that require mechanical intervention to restore PaCO2 to control levels.

Many physiological effects of chronic hypercapnia have been previously reported, and include robust adaptations in acid/base, and respiratory control mechanisms. However, the effects of restoring PaCO2 to control levels following chronically elevated PaCO2 are largely unknown. Thus, the goal of my project is to gain insight into the physiological and ventilatory control adaptations during the acclimatization to‐ and deacclimatization from‐chronic hypercapnia. Based on studies focusing on the deacclimatization from chronic hypoxia, I hypothesize that deacclimatization from chronic hypercapnia would not be completed within 24 hours of return to normoxia.

To assess the effects of deacclimatization from chronic hypercapnia, I measured the temporal pattern of ventilation, ventilatory CO2/[H+] chemoreflex, and acid‐base status during acclimatization to‐ and deacclimatization from‐chronic exposure to elevated inspired CO2 (InCO2) in goats. Goats were chronically housed in environmental chambers that allowed for modification of ambient CO2 levels. Following a room air control period, goats were exposed to an InCO2 of 6% for 30 days, followed by returned to room air for 24 hours (hrs). Within 1 hour of 6% InCO2, VI increased to 322% above control, but decreased slightly to 293% above control after 24 hours. By Day 2 of exposure, PaCO2 increased 15 Torr and changed minimally thereafter. pH decreased by 0.06 units upon initial exposure but was partially compensated by Day 5 due to an 8mEq/L increase in arterial [HCO3−]. Within 4hrs of deacclimatization, VI decreased to 123% above control, and continued to decrease to near control levels by 24hrs. PaCO2 decreased 13mmHg by 24hrs, while pH increased 0.04 units. The CO2/[H+] chemoreflex was assessed by increasing InCO2 to 3, 5, and 7% at 4 and 24 hours of deacclimatization. The chemoreflex was above control at 4 and 24 hours of deacclimatization. The steady‐state ventilation/[H+] relationship was also slightly elevated at 4 and 24hrs.

Taken together, these data suggest that deacclimatization from chronic hypercapnia is nearly complete by 24 hours. Further studies will extend the recovery time to 5 days to see if the deacclimatization is complete. Additional studies will aim to use western blotting to investigate neurological adaptations underlying the deacclimatization process

Support or Funding Information

Department of Veteran Affairs
Sex Specific Changes in SOD1 Proteomics in Acute Ischemic Stroke
Authors: Frank Jacqueline , Maglinger Benton , Roberts Jill , Trout Amanda , Stowe Ann , Grupke Stephen , Pennypacker Keith , Fraser Justin
Published: April 2020

Introduction

Superoxide Dismutase 1 (SOD1) plays an important anti‐oxidant role by destroying free superoxide radicals, and serum levels increase in response to smoking, which is a known risk factor for stroke. The pathology of ischemic stroke includes activation of free radical pathways within the ischemic tissue. Through our previously published Blood and Clot Thrombectomy Registry and Collaboration (BACTRAC) stroke tissue bank (clinicaltrials.gov NCT03153683), we aimed to determine any relationship between intracranial peri‐infarct intraluminal SOD1 proteomics and sex, smoking status, and immediate outcome in stroke patients.

Methods

Plasma samples were processed in accordance with the BACTRAC protocol, and underwent Proximity Extension Assay (PEA) via Olink. For each protein, intracranial expression distal to the stroke thrombus was compared to the same subject’s systemic arterial blood as an internal control. Smoking status, and sex difference were analyzed using appropriate two‐way comparisons and regression.

Results

22 adult subjects (>18yrs) were included in the study, of which 13 (59%) were female. Median age was 64 (24–91). 7 (32%) were active smokers and 5 (23%) had a previous stroke. Mean infarct time was 552 ± 254 minutes and mean infarct volume was 62,361 ± 87,735 mm3. The female population intracranial SOD1 expression was greater in active smokers (p=0.004). No significant change in systemic arterial plasma levels were found in either sex.
Moreover, SOD1 intracranial levels adjusted to systemic controls also heeded a sex specific increase in female smokers (p=0.05).

Conclusions

SOD1 protein expression was found to be significantly increased in only the intracranial distal‐to‐thrombus blood of females based on smoking status. There were no significant findings of SOD1 expression in the male population. These data suggest that changes in SOD1 expression are acutely sensitive to smoking status, but that such changes are particular to women, emphasizing a major difference in how men and women process stroke pathophysiology.

Support or Funding Information

University of Kentucky Center of Clinical and Translational Science
Changes in Morphology of Primary Muscle Cells Exposed to Di‐(2‐ethylhexyl) Phthalate
Authors: Zenteno-Savín Tania , Hernández-Almaraz Pablo , Lugo-Lugo Orlando , Gaxiola-Robles Ramón , Vázquez-Medina José Pablo , Ramírez-Jirano Luis Javier
Published: April 2020

Di‐(2‐ethylhexyl) phthalate (DEHP) is used in plastic manufacture mainly to provide flexibility. Asthma, infertility, endocrine disruption and some types of cancer have been associated with environmental DEHP exposure in humans; however, the morphological, biochemical and physiological changes that lead to these pathologies have not been reported. The objective of this study was to analyze morphological changes in muscle cells exposed to DEHP. Abdominal muscle samples were collected from healthy women subjected to programmed cesarean section at Hospital de Especialidades Médicas de La Paz, in La Paz, Baja California Sur, México, and were subjected to cell culture at 35°C with 5% CO2 and constant humidity. Primary smooth muscle cells (4.0 x 105 cells mL−1) from both species were exposed to 1000 μM DEHP for 13 days. Untreated controls reached 100% confluency, underwent constant mitotic divisions and had normal cell morphology throughout. In contrast, cells exposed to DEHP did not reach confluency beyond 85%, had significantly lower cell viability and total protein content at day 13 of exposure. Cells exposed to DEHP were distended and had granules in the cytoplasm, which were evident since day 2 of exposure and covered all the cytoplasm by day 13. In numerous cells, bright yellow refringent, round bodies were observed in the cytoplasm, with variable size and number among cells. These spheres were observed from day 2 of exposure and are presumed to be DEHP accumulated inside the cells. These intracellular DEHP deposits could be associated with the observed cell mortality and morphology changes and could potentially lead to the reported pathologies onset and progression. This research was conducted in accordance with institutional ethical standards and with the 1964 Declaration of Helsinki and later amendments; informed consent was obtained from all participants included in the study. The informed consent form and research protocol were approved by the Ethics Committee of Instituto Mexicano del Seguro Social.

Support or Funding Information

Consejo Nacional de Ciencia y Tecnología (CONACYT‐Fronteras 2016‐01‐2305)
Building a Virtual Anatomical Library: BlueLinkVR
Authors: Alsup B. Kathleen , Fox Glenn M.
Published: April 2020

The University of Michigan BlueLink project provides expert reviewed and produced anatomical resources, specializing in cadaveric multimedia and curricular materials. BlueLink has begun developing a library of virtual (3D) resources. Using a Shining 3D EinScan‐Pro structured light scanner, osteologic and plastinated specimens are digitized and a 3D scan is produced within EinScan software. Initial scans focus on capturing the gross 3D details of the whole specimen. Once the scan is complete a combination of Meshmixer, Maya, and Blender are used to clean the models and finalize them both structurally and aesthetically. Sketchfab serves as our primary tool for distributing and annotating models. We provide all finalized models as free resources on Sketchfab, which provides an excellent platform to embed resources within our existing BlueLink website.
Characterizing the microbiota‐gut‐brain axis in a murine model of pediatric inflammatory bowel disease
Authors: Stokes Patricia , Salvo Eloisa , Knotts Trina , Rabasa Gonzalo , Sladek Jessica , Gareau Melanie G.
Published: April 2020

Background

Inflammatory bowel diseases (IBD) are chronic intestinal diseases affecting over 1.4 million Americans. Pediatric patients are especially vulnerable to the psychiatric effects seen in IBD as they are still undergoing brain maturation and neuroplasticity during periods of significant GI inflammation. A murine model of pediatric IBD and the mechanism(s) in which the microbiota‐gut‐brain axis is altered during this developmental time have yet to be identified.

Aim

Our goal was to develop a pediatric model of IBD and assess the effect on the microbiota‐gut‐brain axis at adulthood.

Hypothesis

We hypothesized that induction of ulcerative colitis by dextran sodium sulfate (DSS) at weanling mice (P21) would lead to lasting effects on the microbiota‐gut‐brain axis into adulthood, including behavioral deficits.

Methods

C57BL/6 mice (male and female) were weaned (P21) and treated with 2% DSS (or vehicle) for 5 days in the drinking water. In early adulthood (6–8 weeks of age), behavioral tests were performed (novel object recognition task [NOR], light/dark [L/D] box and open field test [OFT]) and tissue samples collected for PCR, immunofluorescence and microbiota analysis.

Results

DSS‐treated mice showed impaired object recognition and increased anxiety‐like behaviors compared to vehicle controls. Hippocampal gene expression showed increased expression of the pattern recognition receptors (Nod1 and Nod2), inflammatory cytokines (IL17ar, IL23 and IL22) along with a decreased expression of the neuronal activation markers (cFOS). Cell proliferation in the hippocampal dentate gyrus as quantified by doublecortin (DCX) staining showed significantly less DCX than vehicle controls. Finally, microbiota assessment identified persistent dysbiosis in DSS‐treated mice on alpha diversity, taxa abundance and phylum abundance compared to vehicle controls.

Conclusions

Taken together, these results indicate that acute DSS‐induced colitis in early adolescence can have long‐lasting effects on the microbiota‐gut‐brain axis in adulthood. Future studies will assess whether administration of probiotics can ameliorate these colitis‐induced microbiota‐gut‐brain axis deficits.

Support or Funding Information

Funding provided by Crohn’s & Colitis Foundation (CCFA)
The CB1 cannabinoid receptor‐mediated inhibition of cAMP accumulation in neuroblastoma cells: role of different Gi/o protein subtypes
Authors: Eldeeb Khalil , Wittmann Thomas G. , Leone-Kabler Sandra , Howlett Allyn C.
Published: April 2020

The CB1 cannabinoid receptor (CB1R) is a G protein coupled receptor with robust expression in the nervous system. CB1R signaling is mediated by pertussis toxin (PTX)‐sensitive Gi /o proteins, and leads to inhibition of adenylyl cyclase. Because of the importance of the cyclic AMP pathway in differentiated neuronal functions, the investigation of G protein subtypes involved in the inhibition of adenylyl cyclase activity is of particular interest. The aim of the present study is to investigate the effect of individual Gi/o proteins on CB1R‐promoted inhibition of forskolin‐ stimulated cAMP accumulation in N18TG2 neuroblastoma cells. We utilized [3H]cAMP binding competition assay and cell membranes in ligand‐modulated [35S]GTPγS binding and scintillation proximity assay (SPA) as previously described (
Blume et al 2015 and
Eldeeb et al 2017).

The CB1R agonist, CP55940 (100 nM) inhibited forskolin‐stimulated cAMP accumulation to 34±8% of forskolin alone. Pretreatment with 100 ng/ml of PTX for 20 hrs attenuated CP55940 induced inhibition to 85±24% of forskolin control. In N18TG2 cells that were stably expressing either PTX‐resistant Gαo, Gαi1, Gαi2 or Gαi3 proteins, treatment of PTX‐pretreated with CP55940 (100 nM) inhibited cAMP accumulation to 38±3% Gαo, 53±4 Gαi1, 76±24 Gαi2 and 28±15% Gαi3 of forsklin alone. Using SPA, CP55940 (100 nM) stimulated [35S]GTPγS binding mediated by Gi/o protein subtypes, Gαo, Gαi1, Gαi2 and Gαi3 in N18TG2 Gαi3 PTX‐resistant cell membranes. In contrast, in PTX‐pretreated membranes CP55940 was able to stimulate [35S] GTPγS binding mediated by Gαi3 only. In conclusion, these data show that Gi/o proteins contribute differently to CB1R‐promoted inhibition of cAMP in N18TG2 cells, with greater contribution by Gαi3. This is consistent with our earlier findings of the high level of Gαi3 expression and its contribution toward total G protein activation in N18TG2 membranes (
Eldeeb et al 2017). The contribution of different G protein subtypes to CB1R signaling is necessary to consider as we evaluate CB1R functions in the nervous system.

Support or Funding Information

NIH grants R01‐DA042157, P50‐DA006634
Electrospun Antimicrobial, Anti‐Inflammatory and Analgesic Bandages for Comprehensive Wound Treatment
Authors: Chirra Bhagath , Shah Kush N. , Chapman Morgan J. , Raina Rohit , Southerland Marie R. , Cannon Carolyn L.
Published: April 2020

Chronic wounds affect more than 5 million Americans annually with an associated cost of $10 – 20 billion. These wounds provide perfect environments for bacterial growth, and are prone to infections, especially from multi‐drug resistant (MDR) pathogens. The current silver‐based bandages used to treat wounds exhibit a high toxicity toward fibroblasts, and lack anti‐inflammatory and analgesic activity. Our work focuses on the development of bandages to address these unmet clinical needs. We have fabricated electrospun polycaprolactone (PCL) scaffolds, capable of aiding in the wound healing process, and with incorporation of Ag+IBU, a novel therapeutic. These scaffolds deliver the drug locally at a controlled rate to impede bacterial infections. We have further functionalized PCL scaffolds with addition of polyethylene glycol (PEG) to accelerate drug release. Scanning electron microscopy images have revealed that our drug loaded scaffolds have a smooth fiber morphology and fiber diameter that is ~1 μm. Scaffolds loaded with either 5% or 10% Ag+IBU (w/w) release 40% of the drug within the first 24 hours. Microbiological assays have revealed that Ag+IBU‐loaded scaffolds are bactericidal against bacteria in both planktonic and biofilm growth mode. Cytotoxicity assays have revealed that Ag+IBU‐loaded scaffolds exhibit toxicity toward human dermal fibroblasts at high concentrations. However, pre‐incubation with N‐acetyl cysteine (NAC), an antioxidant metabolite, abrogates this toxicity and significantly increases cell viability. We have fabricated two‐layer scaffolds with one layer rapidly delivering NAC and the second layer slowly releasing Ag+IBU. Compared with the commercially available bandages with antimicrobial properties currently used for wounds, our non‐toxic, antimicrobial, anti‐inflammatory, and analgesic scaffolds offer a more comprehensive treatment option.

Support or Funding Information

This research is supported by NIH grant 5R21AI142327‐02
Photoaffinity Approach Reveals Antibiotic Adjuvant Activity toward Pseudomonas aeruginosa
Authors: Miller Conrad Laura C.
Published: April 2020

Pseudomonas aeruginosa is an emerging public health threat. A common Gram‐negative source of secondary infections, it has few effective treatment options, which are currently being undermined by the rise of resistance. New therapies are urgently needed. We had previously pursued an antivirulence strategy to block the production of pyocyanin, a redox‐active virulence factor. A photoaffinity analog of an antipyocyanin compound was developed to interrogate the inhibitor’s molecular mechanism of action. In the process, antibiotic adjuvant activity was suggested by the proteomics results. Using susceptibility assays, we found that these compounds amplify the bactericidal activity of colistin, a well‐characterized antibiotic, suggesting they may represent a first‐in‐class antibiotic adjuvant therapy. Analogs have the potential to not only widen the therapeutic index of cationic antibiotic peptides like colistin, but to be effective against colistin‐resistant strains. Multidrug‐resistant infections with P. aeruginosa are currently treated with colistin and the related polymyxin b, however, resistance to these drugs is also increasingly encountered. The adjuvants have the potential to preserve the life‐saving therapy of colistin when used in a combination therapy.

Support or Funding Information

National Institutes of Health 5SC3GM118199

California State University Program for Education & Research in Biotechnology New Investigator Grant
Using S. cerevisiae to screen cancer mutations in the DNA damage response pathway
Authors: Shanle Erin K. , Harris Caitlin , Savas Jessica , Coddington Ashley , Ray Sreerupa
Published: April 2020

When DNA is damaged, a DNA damage response (DDR) pathway is initiated to prevent mutations from being passed on to daughter cells. The MRN complex in humans (MRX in yeast) functions to start the DDR pathway by detecting DNA damage and is comprised of Mre11, Rad50, and Nbs1 in humans. Although mutations in members of the MRN complex pre‐dispose individuals to cancer, the functional effects of Mre11 cancer mutations have not been well characterized. The goal of this project was to investigate the effects of Mre11 cancer mutations on the DDR pathway using yeast‐based assays because the complex is highly conserved in eukaryotes. Using a database of human tumor mutations, we identified 233 cancer mutations in human Mre11, 83 of which occurred at residues that are conserved between yeast and human. A subset of these mutations were screened for functional effects in yeast by creating the cancer mutations at the conserved residues in yeast Mre11. Yeast expressing wild‐type or mutant Mre11 were then assessed for DNA damage sensitivity using hydroxyurea (HU) and methyl methanesulfonate (MMS). Results indicate that N‐terminal point mutations in yeast Mre11 do not confer sensitivity to DNA damage in yeast cells suggesting that these regions may have conserved sequence without conserved function. However, one mutation near the capping domain of Mre11 does show the disfunction of the yeast Mre11 protein similar to the deletion strain. Using yeast‐based assays, cancer mutations can rapidly be screened for functional effects on DDR pathways.

Support or Funding Information

Longwood University Faculty Research and Development Grant
Comparison of Physiological Testosterone Therapy on Androgen Receptor‐Positive and ‐ Negative Cell Lines
Authors: Terrell Nicholas A. , Patel Osman
Published: April 2020

1 in 8 women in the US will be diagnosed with breast cancer in their lifetime. Targeted therapy is a class of treatments that bind to tumor receptors to increase prognosis. However, forms such as Triple‐negative breast cancer (TNBC) do not express estrogen (ER) or androgen (AR) receptors, thus have no targeted therapy. In contrast, Luminal A cancer (LA) does express AR and ER. Recent studies indicate that AR may be a new target for therapy. Therefore, we can identify the role of AR by comparing MDAMB‐232 (AR−) and MCF7 (AR+) cell counts and the percent of AR and ER positive cells at the end of androgen treatment. 2 days before treatment, culture flasks (T25) were seeded with 4×10 5 of MDAMB‐231 and MCF7 cells to acclimate. The cells were then exposed to 0 nM (control), 0.02 nM, 0.2 nM, or 2 nM androgen supplementation for 9 days (n=3). The cell counts were calculated by the Trypan Blue (Gibco) exclusion test. The percent of AR and ER positive cells were calculated using antibodies. In 2 nM androgen MDAMB‐231, cell count was reduced by 48.2%, AR positive cells increased by 52.3%, and ER positive cells increased by 58.8%, compared to the control. In 2 nM androgen MCF7, AR positive cells increased by 14.5% and ER positive cells increased by 16.8%, compared to the control (Statistics p+/−0.05). To conclude, TNBC cells are more receptive to androgen therapy compared to LA cells.
The Search for Novel Splicing Factors: Mapping Mutations in a Temperature‐Sensitive Schizosaccharomyces pombe Library
Authors: Williams Jelani Akil , Dwyer Zachary , Fair Benjamin , Larson Amy , Pleiss Jeffrey A. , Inada Maki
Published: April 2020

Gene expression, starting with the transcription of DNA to messenger RNA (mRNA) and the subsequent translation of mRNA transcripts to form proteins is a vital process for all living cells. Newly transcribed RNAs must be processed before they can serve as templates for protein synthesis. RNA splicing is a key step in RNA processing. Defective splicing disrupts the production of functional proteins and is linked to debilitating human diseases such as cancer, frontotemporal dementia, and Duchenne muscular dystrophy. However, the full set of factors involved in the splicing pathway remain unknown. Hence, uncovering the complete set of splicing factors is critical for the development of new therapeutic interventions.

A recent sequencing‐based genetic screen
1,
2 of a temperature‐sensitive (ts) Schizosaccharomyces pombe library
3 identified a subset of mutants that displayed global splicing defects, which may highlight essential genes with potentially novel implications in splicing. The identities of the specific mutations for each of the strains in this ts library (N = ~2300) have never been previously mapped. Therefore, we aim to map the mutations in this library and quantify their impacts on RNA splicing, allowing us to identify the genes detected in the recent screen and possibly reveal additional novel splicing factors and functional connections. Here we use Tn5 tagmentation for genome fragmentation and Gibson assembly for the high‐throughput synthesis of a wild‐type (WT) cloning library, which will then be transformed into each mutant strain to identify the mutant alleles via complementation testing. Once the alleles have been identified, we will characterize their global splicing deficiencies by sequencing methods to measure the ratio of unspliced to spliced RNA transcripts for all introns. Our efforts will annotate the ts mutant library for general use and contribute to the knowledge of the elements involved in RNA splicing.