bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Dynamic Simulations for Mammalian Target of Rapamycin (mTOR) Variants to Characterize Potential Disruption of Porphyromonas gingivalis Induced Degradation
Authors: Rodin Amanda , Shor Audrey C.
Published: April 2020

Gingipains are a protease secreted by Porphyromonas gingivalis, a major cause of adult periodontitis. P. gingivalis infections are associated with suppressed adaptive immunity and increased neuroinflammation among individuals with Alzheimer’s disease. Gingipains promote neuronal damage and are associated with immunoreactivity. The mammalian target of rapamycin (mTOR) is one protein potentially influenced by gingipain function. mTOR, a protein kinase, controls rates of protein synthesis and degradation, and may also play a significant role in the Alzheimer’s phenotype. Recent evidence suggests that P. gingivalis may inhibit mTOR signaling Alzheimer’s patients. We sought to investigate the role mTOR variants may play in the interaction with gingipains and Alzheimer’s disease. The objectives were to investigate the effect mTOR variants had on structure, function and interaction with gingipains. It was hypothesized that wild type mTOR may confer gingipain induced protection from Alzheimer’s disease, while variant phenotypes may contribute to disease progression. Molecular dynamic simulations were performed to measure hydrophobic collapse and the average movement in 10ns for each structure and compared to wild type. Results suggest structural alterations among the variants which may influence interactions with gingipains. These structural changes may lend evidence to suggest subsequent in vivo analyses investigating how P. gingivalis infection may influence mTOR signaling plays in the Alzheimer’s phenotype.
The Use of a Portable Virtual Reality System as a Supplementary Educational Tool in a First‐Year Human Anatomy Course
Authors: Albabish William
Published: April 2020

Significant advancements in consumer‐grade virtual reality (VR) systems are providing educators with new tools to interact with students. Prior to 2016, VR systems were cost‐prohibitive, limited in software selection, and complex to use. The Oculus Rift, first introduced in 2013, revolutionized the VR field by bringing forth the first affordable six‐degrees of freedom (6‐DoF) commercialized VR system. A functional 6‐DoF VR system consists of the wearable headset, two hand controllers that allow users to interact with objects in the VR space naturally, external sensors to track movements, and requires a costly VR‐ready computer system to render the graphics.

In the fall of 2017, a strike affecting all colleges in Ontario limited the ability to deliver onsite lectures for five weeks. During the strike, innovative ways of lecture delivery, such as VR lectures, were successfully implemented to meet the course objectives of a first‐year dissection‐based human anatomy course. However, a notable limitation of VR technology at the time was its need to be tethered to a computer, which confined its use to a small, pre‐defined work area. The limited accessibility of the technology led to a higher amount of time and effort required to create VR‐based educational content. Recent advancements in VR technology led to a new, all‐in‐one 6‐DoF VR system. The Oculus Quest, released in May 2019, incorporates an on‐board computer, built‐in camera sensors, and an on‐board battery. The combination of these features resulted in a powerful, affordable, and portable system that can be used anywhere, anytime, with ease.

The Oculus Quest was incorporated into the Fall 2019 first‐year human anatomy course. Videos re‐enforcing course lectures and major anatomical concepts, such as abdominal blood supply, the brachial plexus, and blood supply to the upper limb, were created and distributed digitally to students. Additionally, new software features allow the instructor to export the 3D models created in the VR space as a 3D file, which allows students to view the 3D models on their own VR headsets or computer devices, and 3D print the structures created in the VR space. Preliminary data suggests that students had a better understanding of anatomical concepts, relationships, and depth when using the supplementary VR resources in conjunction with traditional resources. Moreover, VR video content attained a higher viewer retention rate and viewer satisfaction when compared to 2D videos.
Analysis of Systemic Transport Barriers for the Activation of Anti‐tumor Immunity
Authors: Nikmaneshi Mohammad R. , Baish James , Padera Timothy P. , Munn Lance L
Published: April 2020

Initiation of an immune response requires activation of one or more naïve T cells. Each naïve T cell has a unique T cell receptor, and these cells traffic through the blood and lymphatic vasculatures, visiting each of the ~500 lymph nodes in search of matching antigen. If a match is made, then the naïve T cell activates and proliferates. The resulting effector T cells enter the blood circulation to find and eradicate the source of antigen. Similar to pathogens like viruses, tumors can also create antigen that accumulates in local lymph nodes, where it can activate T cells. Unfortunately, even when this happens and the anti‐cancer effector cells find the tumor, they are prevented from killing by immune checkpoint programs that quench the immune response. In the past few years, multiple drugs have been approved that block these checkpoint programs, re‐enabling anti‐tumor immunity, and there have been miraculous cures in a subset of patients. However, there is an urgent need to understand why most patients (~85%) do not respond to these therapies. We explore the hypothesis that immune activation in these non‐responding patients is prevented or delayed due to transport limitations caused by the tumor that prevent colocalization of cognate naïve T cells and tumor antigen in the same lymph node. The model provides a mechanistic framework for optimizing cancer immunotherapy and developing testable solutions to unleash anti‐tumor immune responses for more patients with cancer.
Strategies for fostering academic integrity in the classroom
Authors: Parente Amy , Roecklein-Canfield Jennifer
Published: April 2020

Academic integrity can be defined in many ways and can have different implications and applications from both the student and instructor perspective. Following a pilot study gathering data from the authors’ host institutions, data was gathered from faculty attending the summer 2019 ASBMB Transforming Education meeting during a “living poster” presentation on the topic. Specifically, the authors asked questions probing faculty knowledge about cultural norms at their institution surrounding academic integrity and what they perceived were their biggest challenges in the classroom. Faculty responses focused on understanding motivations for student cheating, sharing real‐world examples of unethical behavior with students, and determining strategies for mitigating cheating. These observations led to the development of a survey distributed across the STEM community, from which the data will be used to generate a reference of best practices for fostering academic integrity in the science classroom.
Acute and Chronic Laryngeal Deficits Following Cervical Spinal Cord Injury
Authors: Pitts Teresa , Frazure Michael , Strecker Courtney , Greene Clint , Howland Dena , Iceman Kimberly
Published: April 2020

Laryngeal dysfunction is a common symptom following cervical spinal cord injury (cSCI) and currently there are no effective pharmacological interventions or behavioral treatments. The larynx is the primary valve controlling entrance into the trachea and lungs via opening and closing of the vocal folds. Although not well studied following cSCI, it is understood that laryngeal deficits during swallow often produce aspiration of food/liquid, significantly increasing risk of pneumonia. Our previous terminal electrophysiology experiments using freely breathing sodium pentobarbital anesthetized cats with acute hemisections at the 3rd cervical spinal level (C3) showed immediate alterations in laryngeal activity. These alterations persisted for 4 hours (testing duration). The serotonin (5‐HT1A) receptor agonist 8‐OH‐DPAT was delivered via the vertebral artery at low (3 □g/kg) and/or high (30 □g/kg) doses. This agonist altered behavior‐specific muscle recruitment patterns. In particular, the high dose restored laryngeal activity across breathing and swallow, which could be reversed with the competitive antagonist WAY100635 (0.5 mg/kg). In the current study, we tested if alterations seen acutely in laryngeal activity persist chronically, and if the 8‐OH‐DPAT clinical‐correlate buspirone (Buspar) would produce similar therapeutic results. Adult female cats underwent C3 hemisections and recovered for 4 or 8 weeks. Laryngeal endoscopy was performed weekly under isofluorane (3%) during normocapnia and hypercapnia (5% CO2). Asynchronous vocal fold movement was seen in all animals, defined by unilateral movement delays, and the extent of opening and closing of the glottis became variable. In a subset of animals, oral buspirone (5 mg) was given with food. One hour later, repeat endoscopic evaluation revealed full resolution of laryngeal function with synchronized movements during normocapnia and hypercapnia. Most importantly, with termination of buspirone dosing, laryngeal dysfunction returned. This work shows that the anesthetized preparation effectively identifies upper airway dysfunction early and chronically after C3 SCI, and that it can be used as a pre‐clinical model for novel therapeutic interventions. Results also indicate that buspirone is a high‐priority target for additional investigation as a therapeutic intervention after cSCI.

Support or Funding Information

This work was supported by NIH grants HL 111215, HL 103415 and OT20D001983, the Craig H. Neilsen Foundation Pilot Research Grant 546714, Veterans Affairs Rehabilitation, Research and Development RCSB9249S (DRH), Kentucky Spinal Cord and Head Injury Research Trust, Rebecca F. Hammond Endowment (DRH), and the Commonwealth of Kentucky Challenge for Excellence.
Identifying interactions of a mitochondrial localized ligand activated nuclear receptor and mtDNA interacting proteins
Authors: Waterman Hannah , Keogh Dwight , Bestwick Megan L.
Published: April 2020

Mitochondria are key cellular organelles due to their traditional role in cellular metabolism and energy production, but also for their role in other important signaling and biosynthetic pathways. Because of this it is expected there are many levels of regulation for monitoring and controlling mitochondrial biogenesis. Our aim is to determine mechanisms by which mitochondrial localized ligand activated nuclear receptors act as transcription factors, or other possible rolls, within the organelle by assessing interactions of proteins with mitochondrial DNA (mtDNA) and other mitochondrial proteins. The nuclear hormone receptor superfamily composed of the peroxisome proliferator‐activated receptors (PPARs) play a major regulatory role in basic and complex metabolic function as well as energy homeostasis. The peroxisome proliferator‐activated receptor gamma (PPARγ) is a ligand activated type II nuclear receptor that can translocate to the nucleus and the mitochondria. It is mainly expressed in adipose tissue and is a master regulator of adipogenesis through forming heterodimers with retinoid X receptors. PPARγ regulates fatty acid storage and glucose metabolism, has a role insulin sensitivity, and is responsible for leading to a number of diseases such as diabetes, obesity and atherosclerosis. We are specifically interested in characterizing the role PPARγ plays in the mitochondria and to the extent it is associated with the mtDNA transcription process. We have cloned h‐PPARγ in a pH6HTC vector which contains a His‐tag for recombinant expression in E. coli and purification via affinity chromatography. Our current work is to utilize the recombinantly expressed and purified PPARγ protein in sequence specific DNA binding assays, as well as utilize the purified protein in a human in vitro mitochondrial transcription assay using short DNA templates based on known binding sequences to assess transcriptional output. We are also conducting additional experiments using an mtDNA and protein cross‐linking with immunoprecipitation (mtDNA ChIP‐type) assay that assess specific DNA binding in purified mitochondria.
Interactions of Ionophoric Polyphenols with Human Serum Albumin
Authors: Pichardo Rosemary J. , Zahran Mai , Martinez Alberto , Guevara Johnny , Asim Junaid , Gomez Miguel
Published: April 2020

The distribution of a drug is determined, at least in part, by its interactions with serum proteins. Human serum albumin (HSA), the most abundant blood plasma protein, serves as the most common mode of transport for drugs to their target. In our previous work, we have synthesized and studied important characteristics of a series of ionophoric polyphenols as potential anti‐Alzheimer’s disease agents. As part of our ongoing investigations on the biological activity of these compounds, we are now exploring possible transport mechanisms. In our current work, the interactions of the ionophoric polyphenols with HSA were studied via in vitro and in silico methods. In line with in vitro studies, the interaction of the ionophoric polyphenols with HSA were studied by fluorescence and circular dichroism (CD) spectroscopies to obtain binding affinities, bimolecular quenching constants, theoretical number of binding sites and HSA secondary structural changes. Moreover, in line with in silico studies, a preferred mode of binding was predicted by molecular docking and the predicted systems were subject to a 5 ns MD simulation; consequently, RMSD studies were performed to attest for stability of the systems, MM/GBSA binding free energies were calculated to correlate with experimental binding affinities and per‐residue energy decomposition analyses were carried out to analyze residues critical to binding. Using resveratrol, a polyphenolic compound which has demonstrated strong neuroprotective and anti‐Alzheimer disease properties, as a control, in vitro results showed the formation of adducts (static quenching), one theoretical binding site, binding constants (Ka) ranging 104 to 106 M−1 and only slight secondary structure modifications between HSA and the ionophoric polyphenols. Further, in silico results revealed the ionophoric polyphenols and resveratrol favorably binding subdomain IIA of HSA, forming critical interactions to Trp 214 and Asp451. RMSD studies showed the HSA‐IIA system plateauing after a short time indicating stable binding events; in addition, a Pearson correlation coefficient of R=0.80 between the experimental binding affinity and computational binding free energies showed a strong correlation between in vitro and in silico results. Overall, our results seem to indicate that there is a ionophoric polyphenol‐serum albumin interaction, which could have a potential impact on the use of ionophoric polyphenols as therapeutics for Alzheimer’s disease.

Support or Funding Information

This research used resources of the National Energy Research Scientific Computing Center (NERSC), a U.S. Department of Energy Office of Science User Facility operated under Contract No. DE‐AC02‐05CH11231.
Decentering, Aortic Wave Reflection, and Arterial Stiffness
Authors: LewAllen Sarah E. , Vyas Aditi , Thivierge Grant S. , Morin Brigitte E. , Durocher John J.
Published: April 2020

Decentering, a mindfulness‐related quality, involves an individual’s inherent ability to recognize thoughts as simply psychological stimuli and accept a distant perspective. This includes understanding that thoughts and emotions are transient, rather than permanent associations of the individual. Previous studies have concluded that improvements in anxiety following an MBSR protocol were related to an increased ability to decenter. However, it is currently unknown how the ability to decenter might influence wave reflection and arterial stiffness. Therefore, the purpose of this study was to determine if a higher ability to decenter would be associated with lower arterial stiffness. Twenty‐eight individuals (23±6 years) volunteered to participate in this study. All participants had a BMI less than 30 kg/m2, and reported no history of smoking, taking cardiovascular medication, or having diabetes. Subjects were instructed not to consume alcohol, caffeine, or exercise for 12 hours before, and to abstain from food three hours before testing. We performed applanation tonometry with a SphygmoCor system for pulse wave analysis (PWA) at the radial artery, and carotid‐femoral pulse wave velocity (cfPWV) at the respective arteries to estimate arterial stiffness. Participants also completed an 11‐item decentering questionnaire to objectify their ability to disconnect from their emotions. The Spielberger state‐trait anxiety inventory and the Five Facets of Mindfulness Questionnaire (FFMQ) were also completed. Data were analyzed using IBM SPSS statistical software for a median analysis on the ability to decenter, and for independent‐samples t‐tests. Means were considered significantly different when p<0.05. A higher ability to decenter was associated with a significantly lower aortic augmentation index (AIx, 8 ± 4 vs. −1 ± 3%, p<0.05), while cfPWV tended to be lower in those who scored higher on the decentering questionnaire (p=0.07). Our results in a small cross‐sectional sample indicate that the ability to decenter may be associated with lower cardiovascular risk factors such as AIx. Previous studies with very large sample sizes indicate that there is a moderate correlation between AIx and cardiovascular disease risk factors. Our preliminary results are promising in regard to how the ability to decenter may reduce cardiovascular risk.

Support or Funding Information

This project was partially supported by a National Heart, Lung, and Blood Institute award to John J. Durocher (1R15HL140596‐01).
Differential Expression of Inflammatory Response Genes During Acute High‐Altitude Exposure
Authors: Pham Kathy , Heinrich Erica C.
Published: April 2020

Inflammatory signaling and hypoxia responses are linked via HIF and NF‐kB crosstalk. Upon high‐altitude exposure, the inflammatory profile adapts to the hypoxic environment. However, these evolutionarily conserved inflammatory responses to oxygen limitation likely contribute to pathologies such as Acute and Chronic Mountain Sickness, a progressive fatal disease characterized by excessive erythrocytosis. We hypothesize that pro‐inflammatory gene expression will increase upon acute high‐altitude exposure. We compared the inflammatory profiles in whole blood samples collected in the morning during fasting at sea level and after one night at high altitude (3800 m elevation) in 14 healthy sojourners (4 women, 10 men). Expression of 255 inflammation‐related genes was determined via the NanoString Inflammation panel. 15 genes showed significant differential expression at altitude. These include upregulation of LY96, TGFBR1, HSPB2, IL13, IL9, as well as multiple chemokines (p < 0.0003 for all). Additionally, HIF1A expression was significantly correlated with the expression of multiple genes including TLR4 (R = 0.66, p=0.0003), IL6R (R = 0.68, p=0.001), and IFNG (R = −0.71, p = 0.001). The upregulation of chemotactic proteins provides evidence that acute high‐altitude exposure induces pro‐inflammatory signaling. Furthermore, LY96 encodes MD2, a coreceptor with toll‐like receptor 4, which is upregulated in macrophages via HIF and involved in modulating the inflammatory response to lipopolysaccharide. Therefore, this data suggests that at high‐altitude, hypoxic stress may increase susceptibility to subsequent infection.

Support or Funding Information

This work was supported by the University of California, Riverside, School of Medicine.
Application of Machine Learning Techniques for Testing Sensitivity of Cardiac Dynamics to Arrhythmia Vulnerability Parameters
Authors: Aghasafari Parya , Yang Pei-Chi , Clancy Colleen
Published: April 2020

In this study, we developed a new approach to address the importance of arrhythmia vulnerability parameters for cardiac dynamics prediction in response to autonomic stimulation. We generated various case studies using a series of complex electrophysiology protocols for control versus stimulated single cardiac myocytes by autonomic nervous system (ANS). We collected the “TRiAD” related parameters (triangulation (APD TRI), reverse use dependence (RUD), beat‐to‐beat (bTb) instability of action potential duration and spatial APD dispersion (T‐wave area)). We used collected data to train different machine learning algorithms for classifying control versus ANS case studies. We used artificial neural network (ANN), support vector machine (SVM) with Gaussian kernels and random forest (RF) algorithms and observed that RF outperformed the other two algorithms with higher model accuracy for output prediction. Our results illustrated that bTb instability, APD TRI, RUD and T‐wave area are respectively the most important parameters for our classification problem. We also studied the sensitivity of cardiac dynamics to the listed TRiAD parameters. We observed that increase in bTb instability values leads to higher probability of case study being classified into control group while increase in RUD and TRI values result into higher probability of case study being classified into ANS group. Whereas T‐wave area values illustrated different trend compared to three other parameters, where low values could be misleading in decision making about the group that the case study will belong to and higher T‐wave values mostly belong to ANS classified case studies. Therefore, our study suggests the advantages of data analytics techniques for discoveries that may not have been evident.

Support or Funding Information

This study was funded by National Institutes of Health (Grant OT2OD026580).
Visualizing Arc mRNA transfer using single molecule fluorescence assays
Authors: Little Saffron R. , Walter Nils G.
Published: April 2020

The transfer of genetic material between cells has been implicated in both maintaining neuronal function as well as the proliferation of many diseases and disorders, especially neurodegenerative disorders. Regulation of mRNA transfer through transport and localization is conducted by RNA binding proteins (RBPs), making RBPs responsible for control of gene expression. The activity‐regulated cytoskeleton‐associated protein, Arc, is an important component of maintaining neuronal synaptic plasticity which ensures the proper flow of cognitive information. In Alzheimer’s disease, an increase of Arc protein expression and localization in synaptic activity areas has been observed in compensating for memory and cognition loss. Although Arc is thought to be released from the synaptic cleft through Arc‐formed capsids to transfer Arc mRNA into new target cells, the mechanism of action is unknown. This project uses single molecule fluorescence methods to interrogate the dynamics of Arc allowing for visualization of the potential movement of Arc capsids/Arc mRNA and the transfer of this genetic information in human cells. Preliminary studies of the Arc using single molecule fluorescence in situ hybridization (smFISH) and live cell imaging techniques has shown a static trajectory of Arc diffusion. Further studies of the transport and transfer of mRNA from Arc capsids will allow for an understanding how this machinery works that can be the basis for the development of new drug delivery methods for diseases with RNA targets.

Support or Funding Information

NIH grant number: 1T32GM1320461
Spontaneous Splenic Rupture Case Report: an Approach to Anatomy Importance for Radiological Correlation
Authors: Esteban Roberto Javier Rueda , Pedraza-Rodriguez Juan Daniel , Ramirez Daniela Paola , Perdomo Felipe
Published: April 2020

Spontaneous splenic rupture (SSR) is a rare entity
1, usually associated with hematologic malignancies, infectious processes or inflammatory diseases. Timely diagnosis by ultrasound and/or computed tomography
1 allows early clinical and/or surgical management
2, reducing the mortality
1.

Our aim is to expose, through a clinical case of SSR, the importance of anatomy knowledge and its radiological correlation in medical education.

A 26‐year‐old man, diagnosed with chronic myeloid leukemia (CML) without chemotherapy treatment, attends to the emergency department for 10/10 (VAS) colic pain in the right inferior abdominal quadrant, irradiated to the ipsilateral inguinal region, associated with non‐emetic nauseas, abdominal distention and dysuria. At presentation, heart rate was 80 beats/minute, respiratory rate 18 breaths/minute, blood pressure 123/71 mmHg, temperature 36.0 ° C and SatO2 87%.

Initial workup positive findings were leukocytosis (270.000/mm3), low hemoglobin (11.2 g/dL) and hematocrit (33.3%), thrombocytosis (925.000 platelets), and Hypokalemia (2,96 mEq/L). Abdominal computed tomography showed hepato‐splenomegaly and a poorly defined lesion with irregular borders, infiltration and vascular tracts on the spleen inferior segment. Considerable accumulation of free liquid in the abdominal cavity was present.

After 2 hours the patient reported an increase in pain, associated to abdominal guarding and rebound tenderness. Heart rate of 134 beats/minute, respiratory rate 30 breaths/minute, blood pressure 150/70 mmHg, SatO2 84% and metabolic acidosis (pH 7.0).

Based on the suspicion of hypovolemic shock, an emergency xipho‐pubic laparotomy was performed, finding a 1500cc hemoperitoneum, active bleeding, clots and splenic segments. Complete splenectomy was selected obtaining a surgical specimen (spleen) of 2 Kg, 32 cm in cephalo‐caudal length, and a lower pole rupture. Patient’s postoperative evolution was satisfactory, specific treatment for CML was initiated and he was discharged 14 days after the surgical procedure.

An early diagnosis is crucial for the reduction of morbidity and mortality in patients suffering a SSR. This is carried out through the proper interpretation of diagnostic images. This clinical approach requires a broad body anatomy knowledge, and the capacity to extrapolate it to a bidimensional representation. Several researchers confirm that the inclusion of diagnostic‐images in the anatomy curriculum leads to an early exposure of medical student to these tools, which improves their imaging interpretation and diagnostical skills
2. Finally, the implementation of these diagnostic tools in the anatomy program could lead to better medical practice and, as seen in this case, to a reduction on mortality.
Impact of Immune Function on Aerobic Capacity in Rats
Authors: Baumfalk Dryden R. , Colburn Trenton D. , Horn Andrew G. , Kunkel Olivia N. , Musch Timothy I. , Behnke Bradley J.
Published: April 2020

Background

The use of immunocompromised animals has been indispensable in cancer and immunology research. The use of exercise with these models in these areas of research has increased dramatically over the past decade, with little to no evidence of how immune modification of strains of animals is impacting exercise capacity and/or responses to training. This data is critical for extrapolation to exercise recommendations in humans as the majority of cancer patients do not present with primary immunodeficiency diseases. Recently, observations of lower skeletal muscle oxidative capacity between RNU (NIH Nude, immunocompromised) and Copenhagen rats (immunocompetent) have been speculated to result from possible differences between immune function in animals. The purpose of this study was to determine if there are differences in maximal aerobic capacity, through assessment of V̇o2max, between immune‐competent and ‐compromised animals of the same strain. We tested the hypothesis that immunocompromised animals would have a lower aerobic capacity than their immunocompetent counterparts.

Methods

RNU 316 (NU−/−, T‐cell deficient; n=10) and RNU 118 (NU+/−, no deficiency; n=10) male rats (~4 months old) were obtained from Charles River. Both groups were acclimated to motorized treadmill running at varying speeds (20–40 m/min) for 3–5 sessions before beginning V̇o2max assessment. V̇o2max graded treadmill testing consisted of a 2‐minute warm‐up at 25 m/min at a 5% incline followed by 2 minutes at 35 m/min, and subsequent increases in speed of 5 m/min every minute after until a plateau was seen in oxygen consumption with increased speed, or test termination was required for safety of the animal. Tests were repeated a minimum of two times (3 maximally) per animal to verify maximum values were achieved.

Results

There were no significant differences in body mass (NU−, 347±12 g, NU+, 321±8 g; p=0.17). V̇o2max was lower in NU− vs NU+ animals (80.0±1.5 vs. 88.4±1.9 ml·min−1·kg−1; p<0.01). There was a significant difference in respiratory exchange ratio (RER) at V̇o2max (NU−, 1.05±0.01 vs. NU+, 0.98±0.01; p<0.05), but no difference in CO2 production (NU−, 83.7±1.3 vs. NU+, 86.7±1.5 ml·min−1·kg−1; p>0.05).

Conclusion

This study suggests that immunocompromised animals have a decreased aerobic capacity compared to immunocompetent counterparts. This should be considered when both designing exercise interventions in immunedeficient/compromised animals as well as interpretations derived from such studies.

Support or Funding Information

NIH HL137156‐01A1

ACS RSG‐14‐150‐01‐CCE
Regulation of neutrophil secretion by the lysosomal amino acid transporter, cystinosin
Authors: Carvalho-Gontijo Raquel D. , Johnson Jennifer L. , Catz Sergio D.
Published: April 2020

Neutrophil exocytosis is an important immune response, but secretion must be tightly regulated because exacerbated release of the neutrophil toxic mediators is injurious to the host and mediates inflammation. Here we show that neutrophil exocytosis of azurophilic granules is regulated by the cystine lysosomal transporter cystinosin. Cystinosin localized at azurophilic granules and lysosomes in neutrophils. Isolated bone‐marrow‐derived Ctns−/− neutrophils showed exacerbated azurophilic granule secretion under unstimulated conditions, suggesting dysregulation of a cell‐intrinsic mechanism. The defect was specific for azurophilic granules because secretion of gelatinase granules was not affected. Using phalloidin staining, we show that Ctns−/− neutrophils have decreased F‐actin, suggesting disruption of the azurophilic granule‐specific actin barrier. In addition, phalloidin‐based staining showed increased filopodial protrusion formation in Ctns−/− neutrophils, thus affecting cell morphology. Induction of either actin polymerization or depolymerization by jasplakinolide (JK) or cytochalasin, respectively, followed by stimulation with the bacteria‐derived peptide, fMLF, showed that secretion was still significantly increased in Ctns−/− neutrophils, suggesting mechanistic defects beyond impaired actin remodeling. Using Total Internal Reflection Fluorescence Microscope (TIRFM) and Super‐Resolution Radial Fluctuations (SRRF), we show increased numbers of docked granules at the exocytic actin zone in Ctns−/− neutrophils. Immunofluorescence analysis showed increased colocalization of the small GTPase Rab27a with its effector JFC1 in Ctns−/− neutrophils, suggesting that Rab27a drives the increased exocytosis observed in Ctns−/− neutrophils. This was confirmed by treatment with the Rab27a‐JFC1 binding‐specific inhibitor Nexinhib20 (NEI20), which decreased granule docking and impaired secretion of the azurophilic granule marker myeloperoxidase (MPO) in unstimulated and stimulated Ctns−/− neutrophils. Combined treatment with NEI20 and JK further decreased MPO secretion in Ctns−/− neutrophils suggesting the uncoupling of Rab27a activation and actin depolymerization in Ctns−/− neutrophils. This was confirmed by the observation that the number of docking granules decreases in response to the combined treatment. Blocking ERM (Ezrin/Radixin/Moesin) also decreased the number of docked azurophilic granules at the exocytic active zone and impaired MPO secretion in Ctns−/− neutrophils, suggesting the participation of ERM on azurophilic granule exocytosis. In vivo, we found elevated levels of azurophilic granule cargo in plasma from cystinosin‐deficient mice, and Ctns−/− mice showed increased neutrophilic tissue infiltration, denoting systemic activation. Our data suggest that cystinosin is a negative regulator of exocytosis and that neutrophil‐mediated inflammation may play a significant role in disease progression in cystinosis.

Support or Funding Information

CRFNIH
Inhibition of S‐nitrosoglutathione Reductase During Contractions Slows Recovery of Low‐Frequency Force in Isolated Fast‐twitch Muscle and in Intact Single Myofibers
Authors: Gilmore Natalie K. , Hogan Michael C. , Nogueira Leonardo
Published: April 2020

Nitric oxide (NO) is a free radical produced during contractions in myofibers and has been shown to be involved in the regulation of contractility, glucose uptake and myofiber repair. NO can form S‐nitrosoglutathione (GSNO), a by‐product that is metabolized by GSNO reductase (GSNOR). However, it is not known whether GSNO is produced during fatiguing contractions. The ongoing research tested the hypothesis that GSNOR scavenges the GSNO produced during repetitive contractions to protect contractile and calcium handling proteins from being S‐nitrosylated. To test this hypothesis, the effects of acute inhibition of GSNOR (GSNORi; SPL‐334, 10 μM) on contractility during and after fatiguing contractions in fast‐twitch muscle were examined. Extensor digitorum longus (EDL) muscles or flexor digitorum brevis (FDB) myofibers from both hind limbs of mice (C57BL6/J; 8–9 weeks old) were placed in an experimental chamber for isometric force measurements during electrical stimulation. During muscle recovery from fatigue in the EDL treated with GSNORi (n=6), force developed at low frequencies of stimulation was lower than the force developed in the control EDL (55–70% vs. 80% of pre‐fatigue values after 120 min of recovery, respectively, P<0.05). For FDB myofibers, contractile measurements were taken before and after treatment with GSNORi. The contractile paradigm in single myofibers (n=7) produced a higher decrease in force at submaximal frequencies of stimulation at 120 min recovery (~49%) compared to EDL muscle when GSNORi was absent. When GSNORi was present, the force at submaximal frequencies was not further changed by GSNORi incubation (~51%). Contractile and metabolic S‐nitrosylated proteins were detected in muscles following fatiguing contractions in the presence and absence of GSNORi by using the S‐nitrosothiol resin‐assisted capture (SNO‐RAC) method. Together, these results indicate that GSNOR activity has an important role in protecting contractile function during stimulation patterns that lead to muscle fatigue in EDL muscle.
Innate Immunity in Cutaneous Lupus Erythematosus
Authors: Calderon Irene
Published: April 2020

Background

Cutaneous Lupus Erythematosus (CLE) is an autoimmune skin disorder. The adaptive immune system is involved in the etiology of CLE. CLE lesions respond to inhibition of endosomal toll like receptors, hence supporting other disease mechanisms in its pathogenesis.

Hypothesis

Genes associated with defects in the innate immune system would be differentially expressed in CLE skin compared to normal controls.

Method

Using the Gene Expression Omnibus, gene expression data from skin biopsies of patients with cutaneous and normal controls were compared. Data sets selected included: GSE109248, GSE81071, GSE52417, and GSE95474, including acute lupus, subacute lupus, discoid lupus, and normal skin samples. GEO2R was used to analyze the gene expressions between the two sample groups for each data set. STRING (Search Tool for the Retrieval of Interacting Genes/Proteins), GeneCard, and KEGG (Kyoto Encyclopedia of Genes and Genomes Pathway) analysis were used to identify the interaction and function of specific genes.

Results

Genes associated with the NOD like receptor (NLR) signaling pathway were consistently differentially expressed in discoid lupus skin samples in all data sets (p‐value < 8.74e‐05). Five genes associated with the NLR signaling pathway were found to be upregulated in skin samples of discoid lupus patients in all datasets compared to normal controls. These included: STAT1 (signal transducer and activator of transcription 1), Oligoadenylate Synthetases (OAS1, OAS2, OAS3), and AIM2 (absent in melanoma 2). These five genes are known to be involved with transcription activation, regulation of viral infection, and interferon response.

Conclusion

Genes associated with the NLR signaling pathway are differentially expressed in the skin lesions of lupus patients, specifically discoid lupus, compared to normal controls, supporting the role of the innate immune system in its pathogenesis. Further validation studies using experimental methods such as polymerase chain reaction are needed.
Ethanol Extract of Euonymus alatus Leaf Prevents Scopolamine‐Induced Learning and Memory Impairment in Mice
Authors: Woo Yunju , Lim Ji sun , Oh Jisun , Kim Jong-Sang
Published: April 2020

Our previous in vitro study demonstrated that the ethanol extract from young leaves of Euonymus alatus (so‐called burning bush spindle tree) had antioxidant and neuroprotective activities. In the present study, we examined whether oral supplementation of the extract recovers learning and memory capabilities in a scopolamine‐induced acute hypomnesic mouse model. Behavioral tests showed that spatial learning and memory were improved by the extract treatment in scopolamine‐treated mice. Histological damage observed in the hippocampal region of the scopolamine‐treated mouse brain was attenuated by oral administration of the extract. Oxidative stress markers, including plasma 8‐hydroxy‐2′‐deoxyguanosine and hepatic malondialdehyde increased in scopolamine‐injected mice were decreased by the extract treatment. The expression levels of antioxidant proteins such as heme oxygenase‐1 and nuclear factor (erythroid‐derived 2)‐like 2 in the hippocampal tissue homogenates were decreased in group treated with scopolamine alone while the expression of those proteins was restored by the extract treatment. In addition, synaptic plasticity biomarkers such as neurotrophic factors, including postsynaptic density protein‐95, brain‐derived neurotrophic factor and N‐methyl‐D‐aspartate receptor, were enhanced in the extract‐treated mouse hippocampal tissue compared to the group exposed to scopolamine alone. These findings indicate that Euonymus alatus leaf extract could rescue learning and memory impairment caused by oxidative stress‐dependent hippocampal damage through upregulated antioxidant response and strengthened synaptic function of neuronal circuits particularly in the hippocampus.

Support or Funding Information

This study was funded by the Forest Resources Development Institute of Gyeongsanbuk‐do, Andong, Republic of Korea.
A comparison of mitochondrial respiratory function in adult and fetal sheep pulmonary arteries.
Authors: Hanson Shawn F. L. , Murray Samuel T. , Wilson Sean M. , Blood Arlin B.
Published: April 2020

Neonatal pulmonary hypertension (PH) occurs in approximately 6/1000 term infants. It results from insufficient vasodilation of the pulmonary arteries at birth, although the underlying etiology is not well understood. This increase in pulmonary arterial pressure can lead to severe systemic hypoxia due to redirection of blood flow away from the lungs via ductus arteriosus, resulting in systemic hypoxia. PH is more common in human fetuses exposed to chronic hypoxia during gestation, a phenomenon that is also demonstrated in our high‐altitude fetal sheep model.

There is growing evidence that the mitochondria of pulmonary artery smooth muscle cells (PASMCs) play a key role in establishing vascular tone. Mitochondria are exposed to profound changes in oxygen and substrate availability in the transition from fetus to newborn. To gain a better understanding of the developmental changes in mitochondrial function in PASMCs, we tested the hypothesis that mitochondria from fetal vs adult PASMCs would display different substrate‐dependent rates of oxygen consumption and energy production.

Lungs were surgically removed from adult non‐pregnant ewes or near‐term fetuses following greater than 100 days of residing at an altitude of over 3801 meters using protocols approved by the Loma Linda University IACUC. 4th–6th generation intrapulmonary pulmonary arteries were isolated, and denuded of endothelium. The metabolic activity of the arterial segments was analyzed in a Seahorse XFe 24 Analyzer that measures the oxygen consumption rate (OCR) with values being normalized to the arterial segment dry weight. These experiments were conducted in the presence of selective antagonists to enable determination of proton leak, non‐mitochondrial oxygen consumption, basal respiration, ATP‐linked respiration, maximal respiration, and spare capacity.

Four adults and six fetuses were studied. Compared to adults, fetal PASMCs demonstrated a greater maximal respiratory capacity [813.99 +/− 73.20 (pmol/min) vs. 601.64 +/− 58.09 (pmol/min)] and spare‐capacity [593.28+/− 48.56 (pmol/min) vs. 353.74 +/− 40.39 (pmol/min)] for oxygen consumption while using carbohydrates and amino acids as substrates (p≤ 0.003, ANOVA). Other parameters of OCR were not different between fetuses and adults.

These experiments provide initial evidence that there are maturational changes in mitochondrial function. Ongoing studies are evaluating the contribution of long‐term high altitude exposure to these differences in mitochondrial respiration and their importance to phenotypic and functional aberrations in lung function.

Support or Funding Information

(Supported in part by NIH grant HD083132 (ABB), HD098477 (SMW))
Protein‐DNA Interactome of the OxyR Oxidative‐Stress Transcription Factors in Aliivibrio fischeri
Authors: Castro-Martínez Alexander , Pinto-Sanchez Victoria I. , Rodriguez-Bonilla Laura M. , Flores-Cruz Zomary , Rodriguez-Martínez Jose A.
Published: April 2020

Transcription factors (TFs) are sequence‐specific DNA‐binding proteins that dictate cell function by controlling selective usage of genomic information. One of the largest prokaryotic family of TFs is the LysR type transcriptional regulators (LTTRs). These TFs tend to regulate a diverse set of genes involved in virulence, metabolism, quorum sensing, and oxidative stress. Hydrogen peroxide (H2O2) is a well‐known cell damaging agent, which can lead to oxidative stress, disease, and important beneficial signaling roles. OxyR is a transcription factor with an N‐terminal DNA‐binding helix‐turn‐helix motif and a C‐terminal co‐inducer‐binding domain, that is involved in the defense mechanism against hydrogen peroxide‐induced oxidative stress. Although, different OxyR proteins share functional and sequence similarities across different bacterial species, they respond differently to hydrogen peroxide. The symbiotic relationship between Euprymna scolopes and Aliivibrio fischeri is dependent on the initial colonization survival of the bacteria in an environment rich reactive oxygen species. It has been documented that A. fischeri’s genome encodes two OxyR genes, OxyR1 and OxyR2. Studying OxyR1 and OxyR2 protein‐DNA interactomes will allow us to identify key differences and their possible roles in oxidative stress responses. Currently, we have over‐expressed and purified the full‐length OxyR1 and OxyR2 and their DNA binding domain. Protein functionality has been tested by Electric Mobility Shift Assay (EMSA), and High‐Throughput Systematic Evolution of Ligands by Exponential Enrichment (HT‐SELEX) will be used to determine the protein‐DNA interactomes of the OxyR1 and OxyR2. Genomic targets of OxyR1 and OxyR2 will be bioinformatically predicted once the DNA‐binding specificities are established.

Support or Funding Information

National Institutes of Health SC1GM127231.
Efficiency in Brain Removal Techniques: A Potentially Novel Approach
Authors: Robertson Ellen , Klender Sara , Mueller Caroline , Ferriby Andrew , Roth Alex , Batra Ranjan
Published: April 2020

Dissection of the human brain is an important part of health professional education in the neuroscience curriculum. Therefore, it is critical that adequate protocols are utilized to preserve structures when removing brains from cadavers. Previous studies have looked at approaches to extracting the brain and associated structures from the cranial cavity which would best preserve these delicate structures. However, these methods require both extensive dissection experience and more time than suitable for routine use. The aim of this study was to determine the technique for brain removal that minimized the time required for the procedure while preserving key structures for future educational purposes.

The time and ease for three aspects of brain extraction were assessed: removal of the calvaria, subsequent removal of the brain, and transection of the brainstem. Six dissectors (two faculty, four senior graduate students) with prior experience performed the removals. Each tested three calvaria‐removal approaches – circumferential only, circumferential plus a median cut, and circumferential plus a coronal cut. They also tested three approaches to brainstem transection—superiorly through the foramen magnum, inferiorly below C1 vertebra, or inferiorly after removal of an occipital wedge. The time for each procedure was recorded, and its ease was evaluated on a 5 point scale (1 easiest and 5 hardest). The combination of calvaria and brainstem approaches and the order of completion were randomized across dissectors and the 3 brains extracted by each. At the completion of brain removal, all brains were evaluated by two neuroscience course directors for utility in educational contexts. The evaluators rated structures on a list compiled from the laboratory handouts in the medical neuroscience course.

Preliminary examination of the data suggests that removal of the calvaria using only the circumferential cut or using an additional coronal cut took about the same length of time, whereas performing circumferential and median cuts took somewhat longer. However, the ease of removal was greater when either the additional median or coronal cut was employed. Transecting the brainstem took less than a minute if performed through the foramen magnum or below C1, but it took substantially longer if the occipital wedge cut was executed. The ease of transection was greatest when performed below C1, and least when the occipital wedge was removed. There was no clear difference in overall quality of the brains removed by the various techniques.

While there may be advantages to one or the other of the techniques for preservation of specific structures, it appears that all approaches tested were equivalent in yielding brains that could be used for educational purposes.