bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in Cellular Composition of Dentate Gyrus in Response to Intermittent Hypoxia
Authors: Nallamothu Thara , Castro-Rivera Carolina , Vera Adrianna , Garcia Alfredo J.
Published: April 2020

Adult neurogenesis allows for the continual generation of neurons throughout adulthood. It is implicated in the maintenance of normal hippocampal function and is tightly regulated by oxygen homeostasis. Sleep apnea causes periodic cessations in breathing and leads to intermittent hypoxia (IH), which can impair hippocampal based learning and memory. This ongoing study examines how IH influences neural precursors and remodels the neurogenic niche in the hippocampus. We hypothesize that IH destabilizes the pool of intermediate neural progenitors (INPs) and recruits microglia into the area. Our findings indicate that the number of INPs is reduced by IH. Moreover, while IH does not change the number of microglia in the molecular and granular layers of the dentate gyrus, microglia appear to be uniquely recruited into the neurogenic niche (i.e., the subgranular zone) following IH. These findings suggest that IH‐dependent changes in hippocampal adult neurogenesis may stimulate microglial activity which in turn further impact normal hippocampal function. Such effects may play a significant role in influencing hippocampal physiology during untreated sleep apnea.

Support or Funding Information

This work was supported by NIH PO 1 HL 144454, NIH R01 NS10742101 awarded to AJG, and a grant from The BSD Office of Diversity & Inclusion at The University of Chicago awarded to AJG.
Characterization of effector ion channels that mediate excitatory cholinergic modulation of XII motoneurons in neonatal mice in vitro
Authors: Vo Charles D. , Revill Ann L.
Published: April 2020

Obstructive sleep apnea is a sleep disorder characterized by repeated stops and starts of breathing during sleep. During an apneic episode there is a decrease in upper airway muscle tone, particularly of the genioglossus muscle (primary tongue protruder). Recent data suggest that this sleep specific loss of muscle tone is due to muscarinic modulation of hypoglossal (XII) motoneurons that innervate genioglossus. In adult rats, during REM sleep, activation of muscarinic acetylcholine receptors depresses genioglossus inspiratory output. By contrast, in neonatal mouse brainstem slices, muscarine potentiates inspiratory output when locally applied to the XII motor nucleus. To determine whether these different effects of muscarine are due to postnatal maturation, we first need to determine the effector ion channels that mediate the excitatory response to muscarine in neonatal mice. In individual XII motoneurons recorded from neonatal brainstem slices, muscarinic receptor activation inhibited an apamin‐sensitive small conductance calcium dependent potassium current (SK) and potentiated a hyperpolarized‐activated mixed cation current (Ih). It was hypothesized that both effector ion channels contribute to the net potentiation of inspiratory drive by muscarine in neonatal mice. Therefore, we aimed to determine 1) the effect of inhibition of SK, and 2) the contribution of Ih modulation to the modulation of inspiratory drive by muscarinic acetylcholine receptor activation at XII motoneurons in neonatal mice. Using rhythmically medullary slice preparations from neonatal CD1 mice (postnatal day P0–5), we first investigated the effects of blocking SK on inspiratory burst output. Local application of apamin (30s) to the XII motor nucleus had a net effect of increasing inspiratory burst amplitude (n=3) to 134% of baseline (range 118–143%) or 132% (range 111–169%) of baseline (2 and 20 mM respectively). These data are important to establish the contributions of SK to inspiratory burst amplitude before testing how muscarine may modulate SK during inspiratory bursts. We then confirmed that muscarine had a net excitatory effect on inspiratory burst amplitude in neonatal mice (128 ± 6% of baseline, n = 9). Next, we aimed to investigate if muscarinic excitation is mediated by blocking SK channels. In one P3 preparation, local application of apamin attenuated the muscarinic‐mediated increase in XII inspiratory burst amplitude from 157% to 137%. Lastly, in one P2 brainstem slice preparation, contrary to our hypothesis, blocking Ih with ZD7288 caused the potentiation of inspiratory burst amplitude by muscarine to increase from 130% to 166%. Future experiments will be needed to confirm these results. Our next steps will focus on characterization of developmental changes in muscarinic modulation of XII motoneuron inspiratory burst output.
A Putative Cystathionine Beta‐Synthase Homolog of Mycobacterium smegmatis is Involved in de novo Cysteine Biosynthesis
Authors: Mahato Saroj Kumar , Saini Jasleen , Barabote Ravi Damodar
Published: April 2020

Mycobacteria include serious pathogens of humans and animals. Mycobacterium smegmatis is a non‐pathogenic model that is widely used to study core mycobacterial metabolism. Using M. smegmatis, we are genetically investigating mycobacterial pathways for biosynthesis of cysteine, a vital sulfur‐containing amino acid.

Publishedin vitro biochemical studies had revealed three independent routes to cysteine synthesis in mycobacteria involving separate homologs of cysteine synthase, namely CysK1, CysK2, and CysM. However, in vivo data were lacking. The M. smegmatis genome encodes only a CysM homolog and lacks orthologs for CysK1 or CysK2. However, the genome encodes a putative cystathionine beta‐synthase (CBS) protein that has two domains ‐ an N‐terminal domain that shares weak sequence similarity with CysK1 and a C‐terminal domain that is specific to CBS enzymes. CBS is a metabolic enzyme that catalyzes the conversion of homocysteine to cystathionine in all the three domains of life (Bacteria, Archaea, and Eukarya).

To dissect the roles of CysM and CBS proteins in cysteine biosynthesis in vivo, we generated a series of unmarked gene deletion mutants and gene complementation strains of M. smegmatis and analyzed them phenotypically. We found that neither the ΔcysM nor the Δcbs mutants of M. smegmatis were auxotrophic for cysteine. However, a ΔcbsΔcysM double mutant of M. smegmatis was auxotrophic for cysteine. Genetic complementation of the double mutant using either cbs or cysM genes rescued cysteine auxotrophy. Furthermore, the N‐terminal CysK1‐like domain of the putative CBS was sufficient to rescue cysteine auxotrophy. Thus, our in vivo data implicate a role for the putative CBS in cysteine biosynthesis and also suggest that the protein may have dual functions in mycobacteria.

Multidrug resistant (MDR) strains of M. tuberculosis, the causative agent of Tuberculosis (TB), are becoming a global crisis. Mycobacterial sulfur metabolism has emerged as a vital target for developing novel drugs to treat MDR‐TB. Our findings reveal a potentially new target in mycobacterial sulfur metabolism relevant to strategic development of novel TB drugs.

Support or Funding Information

This project was supported by the Cell and Molecular Biology program and the Department of Biological Sciences at the University of Arkansas, Fayetteville, AR 72701.
A Geometric Morphometric Evaluation of Equid Tooth Shape at Natural Trap Cave, Wyoming
Authors: Grass Andy , Jones Jade , Campbell Anna , Higgins Penny , Meachen Julie
Published: April 2020

Natural Trap Cave is a sinkhole cave located in Big Horn County in northern Wyoming. The cave preserves many Quaternary taxa, including pollen and large carnivorous and herbivorous mammals such as various felids and ungulates. Many of these taxa went extinct in North America following the late Pleistocene megafaunal extinction. The cave was first excavated in the 1970s and 80s by teams from the University of Kansas and the University of Missouri, and more recently by teams from Des Moines University, The University of Adelaide, and The University of Rochester, along with a wide variety of volunteers. Specimens from this cave have been used to elucidate floral change in the area, disentangle ancient food webs, and extend the hypothesized range for Beringian wolves.

Horses are the most common megafaunal group from this site, encompassing two genera: Equus and Haringtonhippus, the “New World stilt‐legged” horse, based on post‐cranial elements and mitochondrial DNA. Some recent studies have shown that horse taxa can be identified by tooth occlusal surface morphology. This study aims to test if isolated equid cheek teeth from Natural Trap Cave can be identified to the genus level, between Equus and Haringtonhippus using geometric morphometrics. 2D landmarks and sliding semi‐landmarks were taken around the periphery of a set of upper cheek teeth, including the areas of greatest curvature at the metastyle, mesostyle, parastyle, and around the protocone and post protoconal valley. Principal component analyses on known specimens do in fact separate Equus and Haringtonhippus, and this difference is statistically sigificant, with the primary difference being the width of the protocone. Additionally, molars and premolars also occupied significantly different areas of morphospace, opening up the possibility of identifying tooth position on unknown specimens as well. With the abundance of isolated horse teeth (either Equus or Haringtonhippus) from Natural Trap Cave, having a reliable means to distinguish between genera will allow us to better study the ecological differences between them and better understand the environment and faunal interactions in late Pleistocene Wyoming.

Support or Funding Information

NSF EAR/SGP 1425059 (Natural Trap Cave Revisited: Ancient DNA, Climate and the Megafaunal Extinction) Julie Meachen, PI
Integrating Authentic Biochemistry Research and Peer‐Reviewed Scientific Literature into the High School Classroom
Authors: D’Ausilio Morgan E. , Taylor Jennifer L.
Published: April 2020

Course‐based undergraduate research experiences (CUREs) have been widely adopted at post‐secondary institutions as a way to engage more students in authentic research. Several CUREs report measurable gains in student outcomes, including technical skills, content knowledge and persistence in science. In light of national initiatives to increase investigation and design in K‐12 classrooms, educators are designing research experiences for students at increasingly early stages of their STEM education. However, there are many challenges to designing and implementing research experiences within a K‐12 setting. At Deerfield Academy and The Pingry School, we have adapted the Biochemistry Authentic Scientific Investigation Laboratory (BASIL) CURE to our upper level STEM curricula. The primary research objective of the BASIL CURE is to determine the enzymatic function of uncharacterized proteins using wet‐lab and in silico investigation. We demonstrate that BASIL is sufficiently flexible to implement in a secondary school setting, either fully or in part. To facilitate planning within a high‐school academic calendar, we have defined six “research phases” of BASIL as benchmarks for our students’ progress. These phases serve as a framework for computational and biochemical analysis of enzyme function, while students gain experience in critical thinking and molecular biology techniques. Within each research phase, students demonstrate their competence by completing a set of defined “deliverables” in which they analyze the data they have collected in the form of a written report or presentation. Beyond the curricular design collaboration between our schools, we also promote virtual student collaboration to provide our students with an avenue for communication of scientific findings to their peers. To augment the research experience and to develop our students’ scientific literacy, we have integrated regular journal club discussions into the research class curriculum. Students demonstrate a high level of engagement in this component of the course, and their comprehension of peer‐reviewed articles is assessed both during and after journal club meetings. Adopting the BASIL curriculum as a part of our secondary school course design has facilitated communication with other educators in the BASIL consortium, a growing community that will prove an invaluable resource for K‐12 educators seeking to establish or expand their research curriculum.
Aluminium Nanoparticles Released by Joint Implants ‐ Quartz Crystal Microbalance with Dissipation Analysis
Authors: Pêkala Jakub Radosław , Pêkala Przemysław Andrzej , Tomaszewska Iwona Maria , Santos-Martinez Maria Jose , Radomski Marek , Paziewski Mateusz Paweł , Pasieka Paweł Melchior , Tomaszewski Krzysztof Andrzej
Published: April 2020

Introduction

The rising popularity of joint prosthetics such as total knee arthroplasty, total disc replacement or total hip arthroplasty has woken serious concern of toxicity of the nanoparticles (NPs) released by the prostheses because of corrosion and wear. The NPs are known to influence blood components, but their impact on platelets has not been yet analyzed. In this study we aimed to assess the effect of Al2O3 (Al) NPs on thrombocytes in vitro with the use of novel quartz crystal microbalance with dissipation (QCM‐D) methodology.

Methods

The several NPs (Al2O3 5nm, Al2O3 10nm, Al2O3 20nm, Al2O3 30nm, Al2O3 50nm and Al2O3 80nm) were tested for their ability to induce platelet activation and aggregation with use of flow cytometry, light transmission aggregometry and QCM‐D. The results of the latter test were confirmed with optical microscopy and transmission electron microscopy.

Results

Significant changes in dissipation (D) and frequency (F) were noted in platelet‐rich‐plasma incubated with all assessed NPs (concentration of 5, 2.5, 1, 0.5 μg/mL), in comparison to controls under flow. All tested NPs, except for the largest (80 nm) ones, induced a significant rise of both F and D, revealing thrombocyte aggregation even at the lowest tested concentration (0.5 μg/mL). Optical microscopy revealed platelet aggregation also for 80 nm NPs. In contrast, transmission electron microscopy of 80 nm NPs showed lack of platelet aggregation, however, the NPs caused formation of several platelets clumps similar to aggregates.

Conclusion

Our study provides evidence that Al NPs influence thrombocytes function in vitro. Moreover, different mechanism depending on the size of NPs has been noted. Smaller Al NPs induce platelet aggregation, whereas large particles penetrate the platelets, damaging them and releasing Ca2+ stores. It is not certain if the latter causes thrombosis in vivo. We believe that future in vivo studies might indicate safe aluminium level for patients with Al‐containing ceramic prostheses as well as establish which concentration would be optimal for revision surgery.
ONC201 and Its Potent Analogues Disrupt Mitochondrial Metabolic Function in Triple Negative Breast Cancer
Authors: Fennell Emily M.J. , Aponte-Collazo Lucas J. , Graves Paul R. , Herring Laura E. , Iwanowicz Ed J. , Holmuhamedov Ekhson , Graves Lee M.
Published: April 2020

Background

ONC201 is an imipridone compound in clinical trials for multiple cancer types, including triple negative breast cancer (TNBC). ONC201 is a promising anticancer therapy whose pharmacological target and function are not clearly defined. We have demonstrated that the target of ONC201 and its potent analogues (TR compounds) is the mitochondrial protease ClpP, consistent with previous reports of ONC201‐induced mitochondrial stress and depolarization in TNBC cells.

Study Objective

The objective of this study is to determine if ONC201 and TR compound treatment result in a loss of mitochondrial protein through activation of ClpP, leading to metabolic disruption in glutamine utilization and mitochondrial function.

Methods

To determine if ONC201 and TR compounds disrupt aerobic respiration, galactose was substituted for glucose in cell culture media. We used galactose‐containing cell culture media to shift cells from anaerobic to aerobic respiration, as galactose is utilized through aerobic respiration. The IC50 of ONC201 and TR compounds was measured with glucose‐ or galactose‐containing media by MTS. MitoPlate assays (Biolog) were performed to identify perturbations in the utilization of 30 different substrates in TNBC cells treated with ONC201 or TR compounds. Proteomic analysis of ONC201 or TR compound‐treated cells was conducted using LC‐MS.

Results

Proteomic analysis revealed a large decline in mitochondrial peptides following ONC201 or TR compound treatment. We also demonstrated that substitution of glucose with galactose reduced the IC50 of ONC201 and TR compounds in TNBC cells. In addition, MitoPlate assays suggest a reduced utilization of TCA cycle substrates and L‐glutamine.

Conclusions

Addition of galactose to cell culture media improved efficacy of ONC201 and TR compounds in TNBC cells. Sensitization and loss of mitochondrial peptides suggest that ONC201 and TR compounds cause loss of mitochondrial protein and reduction metabolic activity. Consistent with sensitization results, MitoPlate data demonstrates a loss of TCA cycle substrate and L‐glutamine utilization following ONC201 or TR compound treatment. Glutamine is known to facilitate both cell proliferation and energy production in cancer cells through protein biosynthesis and the TCA cycle. Proteomic analysis is being used to identify ClpP‐dependent protein loss. Uniformly 13C‐labeled substrates will be used to identify perturbations in energetic and biosynthetic pathways following ONC201 and TR compound treatment. Loss of mitochondrial metabolic activity and glutamine utilization may play a role in ONC201 and TR compound anticancer activity.

Support or Funding Information

NIH Pharmacological Sciences T32 (GM007040), University Cancer Research Fund
Myogenic Activation and Endothelial Function in Cerebral Arteries with Metabolic Disease: Is Myogenic Tone Protective for the Endothelium?
Authors: Halvorson Brayden , Williamson Justin , McGuire John , Frisbee Jefferson
Published: April 2020

While it has been well established that the development of metabolic disease, including hypertension, impaired glycemic control, etc. can be associated with increased myogenic activation of cerebral resistance arteries and arterioles, the impact of this shift in pressure‐induced responses on integrated vascular function remains elusive. Using male obese Zucker rats (OZR) at 17–18 weeks of age, we determined that changes to myogenic activation of ex vivo pressurized middle cerebral arteries (MCA) was best predicted by the severity of elevation in mean arterial pressure and allowed for stratification into three groups (mild, moderate, high degrees of elevation in the slope of myogenic activation vs. intravascular pressure).

While dilator responses to endothelium‐dependent stimuli (e.g., acetylcholine) were attenuated in all MCA of OZR, the severity of the attenuation was correlated with the degree of increase in myogenic activation from normal for that vessel. Treatment of the vessels with the anti‐oxidant TEMPOL significantly improved vascular responses to acetylcholine in the mild and moderate myogenic activation groups of MCA from OZR, but had minimal impact on high myogenic response groups. In contrast, treatment of vessels with the mechanosensitive cation channel inhibitor gadolinium chloride to reduce myogenic activation in OZR MCA to levels comparable to that of control LZR had minimal impact on endothelial function in MCA from the mild and moderate groups, but significantly improved endothelium‐dependent responses in MCA from the high group. Combined treatment of OZR MCA with both gadolinium chloride and TEMPOL resulted in endothelium‐dependent responses being very comparable across groups.

Assays for vascular nitric oxide and PGI2 bioavailability provided correlative evidence that endothelial function, while still impaired in OZR in general, may be better maintained in vessels from animals with a higher myogenic activation than would have otherwise been predicted.

Calculations suggest that the increased myogenic activation in the “high” group may serve to reduce wall tension in response to the elevated arterial pressure and that this may contribute to a mechanism that partially protects endothelial function.

Support or Funding Information

CIHR #389769; NSERC RGPIN‐2017‐04536 and RGPIN‐2018‐05450
Inhibition of stearoyl‐CoA desaturase suppresses growth of pancreatic tumors in vitro and in vivo
Authors: Skrypek Kaitlin , Balog Steven , Asahina Kinji
Published: April 2020

Pancreatic ductal adenocarcinoma (PDAC) is a relatively rare cancer type, but is the fourth‐leading cause of cancer death in the US. Although chemotherapy and immunotherapy have been used for patients with PDAC, the overall five‐year survival rate is 8% and there is an urgent need for effective therapies. Stearoyl‐CoA desaturase (SCD) is an enzyme localized in the endoplasmic reticulum and generates monounsaturated fatty acid from saturated fatty acid by introducing a single double bond. In the present study, we examined the role of lipid metabolism regulated by SCD in PDAC growth and survival using Pdx1Cre; LSL‐KrasG12D mice and human PDAC cell lines. We isolated pancreatic tumors expressing epithelial cellular adhesion molecule by magnetic‐activated cell sorting from the Pdx1Cre; LSL‐KrasG12D mouse pancreas and formed tumor organoids in matrigel. We found that a specific SCD inhibitor (A939572) induces degeneration of the tumor organoids. Interestingly, addition of oleic acid (18:1), but not stearic acid (18:0), rescued the inhibitor‐induced degeneration of tumor organoids. Treatment with the SCD inhibitor increased mRNA expression of unfolded protein response (UPR) markers, such as Atf4, Atf6, Ddit3, spliced Xbp1, and Grp78 in organoids. The increased expression of these genes was reversed by addition of oleic acid, but not stearic acid. Similarly, the SCD inhibitor suppressed the proliferation of PANC‐1, a human PDAC cell line. The SCD inhibitor induced the expression of the UPR markers in PANC‐1 and their induction was suppressed by oleic acid, but not by stearic acid. After injection of the SCD inhibitor to Pdx1Cre; LSL‐KrasG12D mice bearing pancreatic tumors, we observed TUNEL+ cells in pancreatic tumors, but not in normal acinar cells. Our data suggest that the inhibition of SCD causes UPR and cell death in pancreatic tumors.

Support or Funding Information

Pilot project funding from NIH/NIAAA grant P50 AA011999, USC Dean’s pilot project program
(Pro)renin Receptor Antagonism Attenuates High Fat Diet Induced Non‐alcoholic Fatty Liver Disease
Authors: Gayban Ariana Julia B , Worker Caleb J , Anakatumpunya Thananun , Earley Yumei Feng
Published: April 2020

Non‐alcoholic fatty liver disease (NAFLD) represents a diverse array of liver damage linked to obesity, and metabolic syndrome. The mechanisms and the treatment for NAFLD remains lacking; however, recent evidence suggests a critical importance of the (pro)renin receptor (PRR) and the renin‐angiotensin system (RAS) in liver lipid metabolism. Our laboratory recently developed a novel PRR antagonistic peptide PRO20 that blocks the fibrotic signaling of the PRR and the activation of the RAS. We hypothesize that PRR antagonism reverses NAFLD development by preventing the activation of liver RAS and fibrotic signaling. To test this hypothesis, wild‐type male C57Bl/6J mice (n=9–10/group) were fed with either HFD (60% calories from fat) or normal fat chow (NFD, 10% calories from fat) for 6 weeks. Two weeks following diet modification, mice were implanted with a subcutaneous osmotic pump containing either PRO20 or scramble peptide (700 μg/kg/d, 4 weeks). At the end of the 6‐week protocol, liver tissues were harvested and processed for Oil Red O (ORO) and Masson’s trichrome staining for assessment of lipid and collagen accumulation respectively. In scramble peptide treated groups, we found that 6 weeks of HFD induced a significant elevation on liver ORO staining (15.90 ± 1.49 %;) compared with the liver from mice fed with NFD (3.60 ± 1.61 %, p < 0.0001). More importantly, PRO20 (9.91 ± 1.46 %; p = 0.001) significantly reduced the ORO staining compared with scramble peptide in mice treated with HFD. Portal vein collagen deposition was significantly lower in mice treated with PRO20 (1.93 ± 0.60%) compared to the control peptide (4.05 ± 0.60%, p < 0.0211) following 6 weeks of HFD. Mechanistically, we found that PRO20 (1.99 ± 0.62) normalized the elevation of liver mRNA level (fold change) of peroxisome proliferator‐activated receptor gamma (PPARγ) induced by HFD (HFD Scramble peptide: 2.61 ± 0.47 vs. NFD Scramble peptide: 1.13 ± 0.47, p = 0.0037), a key transcription factor regulating lipogenic genes in the liver. In addition, PRO20 prevented the activation of key liver RAS components induced by HFD including the angiotensinogen (1.00 ± 0.62 vs. 6.153 ± 0.62, p < 0.0001), angiotensin converting enzyme (1.03 ± 0.63 vs. 2.91 ± 0.63, p = 0.0344), and angiotensin II type 1a receptor (0.35 ± 0.46 vs. 2.20 ± 0.46, p = 0.0091 value). In summary, our data showed that subcutaneous infusion of PRO20 attenuated the development of HFD‐induced NAFLD and portal fibrosis by regulating liver PPARγ and preventing liver local RAS activation. We conclude that PRR antagonism maybe a new therapeutic target for the treatment of NALFD.

Support or Funding Information

NIH/NHLBI (R01HL122770), American Heart Association (17IRG33370128), NIH/NIGMS (1P20GM130459‐5451)
Dual Oxidase 2 as a Mediator of Podocyte Injury in the Diabetic Environment
Authors: Syed Maaz M. , Abraham Sachin , Ford Bridget Marie
Published: April 2020

Glomerular injury is a prominent pathological feature of diabetic nephropathy (DN). In glomerular epithelial cells, or podocytes, hyperglycemia alters slit diaphragm proteins and causes foot process effacement, apoptosis and cell detachment. Oxidative stress has emerged as an important pathogenic mechanism in the development of glomerular injury in DN. However, the mechanisms by which these factors exert their action remain poorly understood.

We provide evidence that the NADPH oxidase of the Nox family, Dual oxidase 2 (Duox2), is present in cultured glomerular cells, including podocytes. Exposure of cultured podocytes to high concentrations of glucose (HG) elicited a rapid upregulation of Duox2 protein expression. Inhibition of Duox2 with specific siRNA prevented the HG‐induced increase in intracellular reactive oxygen species (ROS) generation and hydrogen peroxide (H2O2) production in cultured podocytes.

In additional experiments, we established a functional link between Duox2‐derived ROS generation and podocyte injury in response to HG. Impairment of Duox2 function nearly abolished HG‐mediated DNA fragmentation, apoptosis and decrease in/reorganization of slit diaphragm protein expression in podocytes, indicating that Duox2 is required for the deleterious effects of glucose in podocytes.

Our study demonstrates, for the first time, that Duox2 is responsible for increased ROS generation and subsequent alteration of podocyte function in response to HG. Our work serves as proof of concept to demonstrate the utility of targeting Duox2 as a future therapeutic intervention to reduce diabetes‐mediated glomerular lesions.

Support or Funding Information

This project was funded by the University of the Incarnate Word, Office of Research and Graduate Studies and the following grants: NRSA 5T32HL007446‐32
Epidural Stimulation Targeting Lower Urinary Tract Function in Spinally Intact and Transected Urethane‐Anesthetized Rats
Authors: Hubscher Charles , Hoey Robert , Medina-Aguinaga Daniel , Khalifa Fahmi , Zdunowski Sharon , El-Baz Ayman , Herrity April , Harkema Susan
Published: April 2020

Bladder management for conditions such as spinal cord injury (SCI) includes a combination of catheterization (either self‐intermittent or suprapubic) and pharmacological approaches, which targets maintenance but does not promote recovery of function. We have exciting data from multiple individuals receiving spinal cord epidural stimulation (scES) that is optimized for stepping and standing, in combination with task‐specific training, that shows improvements in lower urinary tract function. The objective of our current functional mapping pre‐clinical animal studies is to identify scES configurations (electrode location, amplitude and frequency) that can promote optimal neural control of urine storage (adequate bladder capacity at safe pressures without incontinence) and sufficient controlled emptying (high voiding efficiency at safe leak point pressures). Initial mapping was done in acute anesthetized terminal preparations during cystometry (bladder filling) using a modified Medtronic Specify 5‐6‐5 epidural array that is being used by our group in parallel human scES studies. The current data compares the response to scES in spinally intact and transected male and female rats 6 weeks post‐injury at T13‐L1 (level of sympathetic supply), L3‐4 (region with circuits related to external urethral sphincter control) and L6‐S1 (level of parasympathetic supply) spinal segments. Results indicate that scES storage effects and voiding efficiency were dependent upon presence/absence of spinal transection, frequency and intensity stimulation patterns, electrode location, and sex of the animals. Taken together these results suggest that the circuitry is dissociable and amenable to stimulation that seeks to promote either storage or voiding. Furthermore, our innovative approach and novel application of this Medtronic Specify 5‐6‐5 epidural device will generate functional maps that will lay the groundwork for expedient translation of this promising technology to individuals with SCI as well as provide a powerful tool for elucidating underlying circuits present in the lumbosacral cord that are involved in urogenital function.

Support or Funding Information

NIH SPARC OT2OD024898
Lactate stimulates central respiratory motor output in bullfrogs, Lithobates catesbeianus
Authors: Burton Michael T. , Santin Joseph M.
Published: April 2020

Ventilation must constantly change to meet varying metabolic and acid‐base regulation needs of the organism. Although O2 and CO2/pH sensors have received much attention for the chemical control of breathing, metabolites such as lactate and ATP have recently been shown to play important roles in both the hypoxic and hypercapnic ventilatory responses of vertebrates. In mammals and fish, lactate anions increase ventilation through peripheral mechanisms, but the actions of lactate in the central respiratory network of any vertebrate species remain unclear. Anuran amphibians (frogs and toads) accumulate large amounts of lactate in the brain during periods of high respiratory drive, suggesting that lactate may act at central sites to stimulate breathing. For example, hypoxia increases brain lactate which may contribute to elevated ventilation following physiological hypoxia caused by breath‐holding. Furthermore, anurans perform mainly anaerobic exercise, leading to large lactate loads in the blood that may get delivered to the brain through transporters present in the blood‐brain barrier. Thus, we tested the hypothesis that lactate stimulates output from the central respiratory network in amphibians. To test this hypothesis, we measured respiratory‐related cranial nerve X (CNX) and hypoglossal nerve (SNII) activity in isolated brainstem‐spinal cord preparations from the American bullfrog, Lithobates catesbeianus, in response to sodium lactate (lac−; n=8). The brainstem‐spinal cord preparation is advantageous because we can assess the actions of lac− on rhythm generating and motor functions of the respiratory network independent of peripheral feedback. Lac− failed to increase frequency of the respiratory rhythm and amplitude of CNX output. However, we identified a striking dose‐dependent increase in amplitude of respiratory‐related SNII output in each preparation, indicating enhanced motor neuron firing and/or recruitment by lac− (P=0.008; Friedman test; 0.5 mM= 33±11, 2 mM= 56±19, 4 mM= 89±29 % increase above baseline). To control for slight increases in [Na+], osmolality, extracellular pH, and time during exposure to sodium lactate, we superfused a separate group of preparations with 0.5, 2, and 4 mM sodium bicarbonate (HCO3−; n=4). HCO3− did not increase the motor amplitude of either SNII or CNX (P>0.9; Friedman test). Thus, lactate anions lead to increased respiratory motor output of SNII, which may enhance activation of buccal floor musculature during periods of high respiratory demand such as hypoxia and exercise. Overall, this work directly implicates lactate anions in the control of central motor networks that regulate breathing in vertebrates.

Support or Funding Information

This work was funded by start‐up funds provided by UNC‐Greensboro to JS.
The Size and Shape of the Alveolar Process: A Morphometric Analysis of the Dental Arch During Fetal and Infantile Development
Authors: Cusick Abbey Marie , Zdilla Matthew J.
Published: April 2020

The development of the alveolar process is closely tied to numerous oral pathologies, including cleft lip and cleft palate. Despite the importance of the alveolar process, little gross anatomical study has been performed with regard to its development. Therefore, this study analyzed 187 dry maxillae from 109 individuals aged from five‐months intrauterine to five‐months post‐natal. Distances between the centers of the five alveoli were measured via photogrammetry. Interalveolar distances and age (months development) were utilized to develop best‐fit regression equations. Paired‐samples t‐tests were performed between left‐ and right‐sided interalveolar distances. Also, geometric morphometric analysis was performed and included canonical variate analysis to identify shape change among different age groups and between‐block partial least squares analysis to assess covariation in the premaxillary alveolar process and alveolar process of the maxilla proper. The interalveolar parameter most closely related to age was that of the intermolar distance (Distance = [−0.02962(age2)] + [0.8854 (age)] + 0.7475; r2=0.5983). Additionally, three out of four inter‐alveolar distances in addition to the total length of the dental arch significantly differed between the left and right sides (p=0.0003, p=0.0027, p <0.0001, p=0.0078, respectively). Alveolar arch shape was significantly different among most age groups; this was especially the case when there was a greater difference in age (i.e., early intrauterine vs late post‐natal). Furthermore, PLS analysis demonstrated a strong relationship between the shapes of the premaxilla and maxilla proper (Correlation coefficients of PLS1 and PLS2 were 0.73 and 0.70, respectively). The results of this study improve the understanding of alveolar process growth― age is best estimated by intermolar distance (and vice‐versa), there is asymmetry between left‐ and right‐sided maxilla growth, dental arch shape changes with age, and the shape of the premaxilla is closely related to the shape of the maxilla proper.

Support or Funding Information

Funding: WV Research Challenge Fund [HEPC.dsr.17.06]
Optimizing Protein Function for Screening of Novel Inhibitors that Target Multi‐Drug Resistance in Cancers
Authors: Okwuone Dakota D. , de Oliveira Maisa C. , Jensen Madeline K. , Vogel Pia D. , Wise John G.
Published: April 2020

P‐glycoprotein (P‐gp) belongs to the ATP‐Binding Cassette (ABC) transporter family of proteins and is strongly linked with multidrug resistance (MDR) in tumors where overexpression often abolishes the effects of chemotherapeutics by removing the therapeutics from the cells. In hopes of creating co‐therapeutics to re‐sensitize tumors to chemotherapeutics, we previously used high‐throughput in silico ligand docking studies and identified drug‐like compounds that reversed MDR in human cancer cells in culture. To evaluate the mechanism of inhibition in human P‐gp, we have isolated human P‐gp from the yeast Pichia pastoris using mixed detergent micelles. In order to effectively assess the potential inhibitors, we must be able to sufficiently stimulate our protein and have a high enough activity to see the inhibitory effects of the potential inhibitors. In previous work, we found that the activity of P‐gp assembled into membrane nanodiscs had significantly higher basal activity in the absence of transport substrate than P‐gp in detergent micelles. However, significant increase in activity in the presence of the substrate verapamil was not observed in micelles or nanodiscs. Here, we report methods of protein purification and nanodisc assembly modified to have optimally functional human P‐gp. The resulting purified P‐gp will be used to evaluate the inhibition mechanism of lead compounds that were shown to reverse MDR in cancer cell culture.

Support or Funding Information

This work was supported by NIH NIGMS [R15GM094771‐02] to PDV and JGW, SMU Hamilton Undergraduate Research Scholars and Undergraduate Research Assistantship Programs, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and private gifts from Ms. Suzy Ruff of Dallas, Texas, and Ms. Myra Williams, Ph.D. of Naples, Florida. The authors would like to thank Dr. Ina L. Urbatsch, Texas Tech University Health Science Center, for providing the P. pastoris cells expressing the human wild‐type MDR1 used in this work.
Diaphragm Muscle Weakness Contributes to Ventilatory Deficits in an Animal Model of Congenital Diaphragmatic Hernia
Authors: Fogarty Matthew J. , Enninga Elizabeth A. L. , Ibrogba Eniola R. , Ruano Rodrigo R. , Sieck Gary C.
Published: April 2020

Congenital diaphragmatic hernia (CDH) occurs in approximately 1 in 2,500 live births; it is associated with high rates of neonatal mortality, especially in cases with severe pulmonary hypoplasia and pulmonary arterial hypertension. Treatments have focused on hernia repair and ameliorating lung hypoplasia, with the precise pathophysiological mechanisms of CDH remain unknown. We investigated whether or not diaphragm muscle (DIAm) weakness is apparent in CDH, evaluating DIAm force generation and ventilatory function in a Nitrofen rat model. 7 pregnant rats received 100 mg of Nitrofen in 2 mL of olive oil at 9 days gestational age and rat pups were collected at birth. Ventilatory function was assessed in isolated plethysmography chambers (Buxco). Following these functional assessments, neonatal pups were euthanized, and the DIAm with its rib attachments excised. The presence or absence of a CDH was confirmed and a DIAm strip was obtained either from control pups, or from the unaffected side in CDH pups. The isometric force generation of these DIAm strips and their fatigueability was assessed using field stimulation of muscle at various frequencies (5–100 Hz) and normalized to DIAm cross‐sectional area. Statistical significance was determined using a Student’s t‐test or Mann‐Whitney u test at the P<0.05 level. A marked reduction in both neonatal respiratory rate (−42%) and respiratory minute ventilation (−43%) was observed in CDH pups compared to controls (P<0.01). We observed reduced DIAm twitch (−40%), maximum tetanic (−72%) and residual force following fatigue (−69%) in CDH pups compared to controls (P<0.05). CDH neonatal rat pups exhibit DIAm weakness and ventilatory disturbances. Based on previous modelling of ventilatory function, it is possible that DIAm weakness contributes to the poor ventilation, as CDH DIAm forces following fatigue were less than control groups. This work has important considerations when assessing novel treatment outcomes in clinical CDH.
Modulation of Airway Remodeling by PF543, a Sphingosine Kinase 1 Inhibitor, in a Mouse Model of Bronchopulmonary Dysplasia
Authors: Sudhadevi Tara , Ha Alison W. , Ebenezer David L. , Fu Panfeng , Putherickal Vijay , Ackerman Steven J. , Kanteti Prasad , Berdyshev Evgeny , Natarajan Viswanathan , Harijith Anantha
Published: April 2020

Bronchopulmonary dysplasia (BPD) is a chronic lung disease affecting extreme preterm newborn survivors with serious long‐term sequela of wheezing. Oxygen therapy, which is inevitable for the survival of such extremely premature infants, is partly responsible for the development of BPD and associated airway remodeling (AWRM). Altered Sphingosine‐1‐phosphate (S1P)/Sphingosine kinase 1 (SPHK1) signaling have been implicated in the pathophysiology of a number of lung diseases; however, the role of SPHK1/S1P signaling in BPD and AWRM is not well understood. The neonatal mice were subjected to hyperoxia (HO) at 95% oxygen (postnatal day 3 to 10) and allowed to grow as adults for 8 weeks. During exposure to room air (RA) or HO, the mice received 5mg/kg of PF543, a specific inhibitor of SPHK1, intraperitoneally on alternate days. Lungs were then collected and prepared for histology as well as morphometry of alveoli/airways. Lung function studies were performed using a ventilator and methacholine challenge was used to measure airway hyper reactivity (AHR). Levels of S1P and sphingoid bases in bronchoalveolar lavage (BAL) fluids and lung tissues were measured by LC‐MS/MS. In vitro mechanistic studies on HO‐induced AWRM and the protective effect of PF543 were performed using small airway epithelial cells (SAEC).

The exposure of neonatal mice to hyperoxia resulted in AWRM in adults, as evidenced by increased peribronchial fibrosis and smooth muscle hypertrophy. These changes were significantly less in Sphk1 knock out (Sphk1−/−) mice exposed to HO indicating a potential role of SPHK1 in the pathogenesis of AWRM. Subsequent experiments using a specific SPHK1 inhibitor, PF543, during neonatal HO prevented alveolar simplification and, more significantly, ameliorated AWRM in adult mice. Lung function studies revealed AHR in adult mice that were exposed to neonatal HO. Interestingly, PF543 therapy reduced methacholine‐induced AHR and airway constriction along with a reduction in AWRM. Exposure of neonatal mice to HO showed epithelial mesenchymal transition (EMT) ‐like changes, which was reduced by PF543 treatment. Inhibition of SPHK1 with PF543 in SAECs ameliorated HO‐induced loss of E‐cadherin and gain of N‐cadherin and vimentin, markers of EMT.

In conclusion, our data indicate a novel role for SPHK1/S1P signaling in HO‐induced BPD and AWRM, and amelioration by PF543.

Support or Funding Information

This work is supported by NIH/NICHD R01 HD090887‐01 A1 and AHA 18TPA34230095 to AH.
Exposure to adropin improves insulin‐induced dilation in arteries from type 2 diabetic mice
Authors: Jurrissen Thomas J. , Fujie Shumpei J. , Ghiarone Thaysa , Ramirez-Perez Francisco I. , Lemitsu Motoyuki , Maeda Seiji , Manrique-Acevedo Camila , Martinez-Lemus Luis A. , Padilla Jaume
Published: April 2020

Vascular insulin resistance is a hallmark of type 2 diabetes (T2D) and blunting of insulin‐induced dilation is its primary manifestation. Importantly, in T2D, reduced insulin‐induced dilation and blood flow to skeletal muscle significantly limits glucose uptake and contributes to impaired glucose control. However, the mechanisms underlying vascular insulin resistance in T2D remain largely unknown. Adropin is a peptide primarily secreted by the liver and brain, recently shown to regulate energy homeostasis as well as to promote systemic insulin sensitivity. Notably, circulating levels of adropin are decreased in T2D, which may contribute to impairments in vascular insulin signaling. Herein we tested the hypothesis that arterial exposure to adropin improves vascular insulin sensitivity, particularly in T2D. To test this hypothesis, femoral arteries from ~12‐week old db/db (n=5) and genetic control (db+/−, n=5) male mice were incubated with adropin (50 ng/mL) or vehicle for 24 hours and vasomotor responses to acetylcholine, insulin, and sodium nitroprusside (SNP) assessed thereafter via pressure myography. Consistent with our hypothesis, we show that treatment of femoral arteries from db/db mice with adropin improves insulin‐induced dilation (P<0.05), an effect that is not observed in db+/− control mice (P>0.05). Adropin exposure did not alter acetylcholine and SNP‐induced dilation of femoral arteries in either db/db or db+/− mice (P>0.05). These findings support the notion that adropin deficiency may contribute to vascular insulin resistance in T2D.
Beyond OIRD: The Multiple Negative Effects of Systemic Fentanyl in Adult Goats
Authors: Buchholz Kirstyn , Neumueller Suzanne , Burgraff Nicholas , Pan Lawrence , Hodges Matthew , Forster Hubert
Published: April 2020

Opioids are effective analgesics but are addictive and negatively impact multiple physiologic functions. Opioid abuse has reached epidemic levels and led to an exponential increase in deaths and societal costs, with a disproportionate impact on women. A majority of studies have focused on the depressant effect of opioids on breathing frequency. However, there are data suggesting that opioids cause multiple life‐threatening physiologic effects, but there is a paucity of data assessing the multiple deleterious effects simultaneously in freely behaving mammals. Here we tested the dose‐dependent effects of systemic fentanyl (a major cause of the current opioid crisis) in adult female goats. Systemic fentanyl acutely and dose‐dependently depressed breathing frequency through prolongation of expiratory time while increasing tidal volume similar to effects of fentanyl in humans (J Clin Monit Comput 18: 2004; Eur J. Anest 20: 2003; Br J. Anest 96: 2006). Fentanyl also increased the difference between alveolar and arterial PO2 (A‐a oxygen gradient,) and at high doses increased tonic activity of intercostal and abdominal muscles (consistent with “wooden chest”) while also prolonging post‐inspiratory muscle activation and in rare instances caused obstructive apnea. Beyond these acute (<15 min) effects were prolonged increases in body temperature, metabolic rate, and arterial blood pressure and decreases in heart rate. Furthermore, high dose opioids had long‐lasting effects, including induction of acute withdrawal symptoms, and led to decreased fecal output for >24 h. We conclude that a triad of deleterious effects on cardiorespiratory responses (respiratory frequency depression, A‐a oxygen gradient, and sustained contraction of respiratory muscles) conceivably contribute to hypoxemia induced opioids fatalities. Furthermore, opioids have profound, deleterious effects (withdrawal and constipation) beyond cardiorespiratory control.

Support or Funding Information

Department of Physiology

Zablocki VA Medical Center