bluuebunny/crossref_metadata_2025 huggingface dataset:
SPARC: Fecobionics Simulated Defecations in Pigs
Authors: Wang Mengjun , Gregersen Hans , Sun Daming , Han Ling , Kassab Ghassan
Published: April 2020

Objective. We have developed an integrated Fecobionics device to assess pressures, orientation and bending simultaneously during defecation. This study assessed the feasibility of the Fecobionics device in pigs.

Methods. Ten Yorkshire pigs (male, 50–60kg) were divided into groups that was euthanized immediately after the experiment (acute group, n=5) or lived for 2 weeks before the pigs were euthanized (chronic group, n=5). Fecobionics is a bendable probe that contains pressure sensors, motion processing units (MPUs), and electronic circuits. The probe was 13cm long, 13mm OD and contained a bag for distension. The pigs were lightly restrained in a sling. The device was inserted manually through an anoscope into the rectum within half hour after the pig had a normal defecation. The bag was filled with 20–50 ml of saline to provide an urge to defecate. Behavior and signs of discomfort was evaluated during and after the defecation. Pressures, orientation and bending data were collected during defecation. Both the rectum and the anal canal were excised after euthanization for mucosal gross morphological observation and histological examination. Devices were tested after the expulsions for any damage to the electromechanical system, the core and bag.

Results. The pigs paid little attention to device insertion if they were distracted by food. Often the pigs became a bit agitated after eating and defecated the device at this point. The device was defecated from few minutes up to 1h after insertion (average 12min). The pressure sensors at the two ends and inside the bag showed simultaneous pressure increase when the pigs contracted the abdominal muscle. The defecation pressure was usually in the range 80–100cmH2O if the pig was not agitated. Device bending was less than 20 degrees during defecations, indicating that the pig does not exhibit an anorectal angle like humans. Fecobionics was slowly pushed out in 10–40 seconds, often with the front hanging from the anus for some time before being dropped. The recovered devices had intact surfaces without exposed electronics. In two cases the bag had a small leak. One device stopped working after being defecated and had a broken wire, likely due to damage when it dropped to the floor. Mucosal damage, bleeding and inflammation were not found in gross morphological observation and histological examination in the acute or chronic groups.

Conclusions. Fecobionics is a safe device providing novel defecatory measurements under physiological conditions.

Support or Funding Information

NIH SBIR phase 1 grant R43DK112543 and NIH SPARC OT2OD025308
Analyzing the effects of past, present, and future water consumption through analog mapping
Authors: Arellano Denisse , Wang Zhihua
Published: April 2020

The average amount of water usage per capita is 70 gallons per day. Individuals do not often consider their daily water consumption. Model simulations are useful in predicting future water consumption scenarios. Simulation models consist of the different relationships of elements and their correspondence to one another. On the other hand, analog mapping is being used more frequently to visually express and examine the differences in data throughout multiple physical geographical locations. In this study we determine the similarities between water usages across various cities. The cities that are compared are along the continental United States of America regardless of their climate variability. Historical water usage from 50 years ago, water usage habits as the current phenomenon, and projected water usage scenarios in a future 50 years from now are carefully analyzed. This study combines water usage predictions along with analog mapping to evaluate water‐availability and water‐usage. Results from this study can be used to assess the impact of unregulated water usage and the risks of water depletion. The interconnection of both water simulation models as well as analog mapping can help prompt distinct water usage perspectives in addition to alternative methods of communication. Further research can help evaluate alternative development scenarios.

Support or Funding Information

NSF LEAP Scholars Program
Mechanisms underlining excitatory synaptic plasticity in the Nucleus Tractus Solitarii following Cardiovascular Deconditioning
Authors: Lima-Silveira Ludmila , Hasser Eileen M. , Kline David D.
Published: April 2020

The nucleus tractus solitarii (nTS) is a critical central region for autonomic control and integration of cardiovascular reflexes. Neuroplasticity in nTS contributes to several cardiovascular dysfunctions. Our previous data showed that Hindlimb‐unloading (HU), a well‐established model of cardiovascular deconditioning in rats, increases glutamatergic neurotransmission yet reduces the excitability of nTS neurons. In this study we aimed to investigate the mechanisms underlining these alterations. For this purpose, stainless wire rings were placed in the tail of male Sprague‐Dawley rats (3 weeks). After one week of recovery the rings was connected to a suspension apparatus and rats were suspended at an angle of 30–35° for two weeks with free access to food and water. Control animals were maintained in normal postural conditions. After the HU protocol, coronal brainstem slices were generated and the whole‐cell patch clamp technique was performed to evaluate the synaptic neurotransmission and electrophysiological properties of nTS neurons. Compared to control rats, HU increased the amplitude of excitatory postsynaptic currents evoked by afferent tractus solitarii stimulus (TS‐EPSC). The addition of a NMDA receptor antagonist (AP5, 10 μM) significantly reduced TS‐EPSC amplitude in HU but not controls. Despite the increase in glutamatergic inputs, the neurons of HU vs control rats are hyperpolarized and have decreased excitability. The block of both NMDA and non‐NMDA (NBQX, 10 μM) glutamatergic receptors did not affect these parameters. However, in the presence of a GABAA receptor antagonist (Gabazine, 25 μM) the resting membrane potential depolarized and excitability of HU neurons were comparable to neurons from control rats. The results indicate that HU increases TS‐EPSC amplitude via a NMDA receptor component. Furthermore, the decreased excitability and hyperpolarized membrane is associated with enhanced GABAergic modulation to these neurons. This functional neuroplasticity in the nTS may contribute to the cardiovascular deconditioning induced by HU.

Support or Funding Information

HL132836 AAS lundbeck fellowship
Experience‐based Modified Sheet Plastination Protocol
Authors: Espinosa-Montagut Nathally , Clavijo-Ordoñez Laura Daniela , Rueda-Esteban Roberto Javier
Published: April 2020

Specimen preservation has been perfected over time through the application of multiple preservation techniques such as sheet plastination. Already described advantages of this technique are: reduction of risks associated with manipulation, longer shelf life and visualization of both internal and external anatomical structures through the sheets.

This method represents an alternative to create models that can be used to teach Gross Anatomy especially because the final cuts obtained from this technique are similar to those seen in diagnostic images such as Magnetic Resonance Imaging or Computed Tomography.

Our objective is to present a modified standardized sheet plastination protocol based on experience gained through the technique development in our laboratory and other previously released protocols.

As a basis, published protocols such as: BIODUR; University of Murcia, Spain; Dalian University, China; National University, Colombia; among others were used.

Since dehydration was the step with the greatest variation, three alternatives were tested: isopropyl alcohol, acetone and ethanol. Forced impregnation is done using BIODUR P‐40 resin until −15mmHg are acquired in the impregnation chamber and cayalysis is then pursued through vertical positioning of the sheets in a Near UV spectrum (NUV) light chamber.

Using acetone or ethanol in addition to a longer dehydration time, requires augmented costs and reactant usage, algo, tissue sheets do not set in the center of the vertical chamber, which resulted in broken or bent sheets. Due to this, isopropyl alcohol was chosen as the best intermediate medium. Dehydration changes were made every three days with the solvent at −20°C, starting with a concentration of 95% and making the last refill at 99.8%. The, specimens were placed in a vacuum chamber with BIODUR P‐40 resin. Constant bubbling was monitored while the pressure decreased until bubbling stopped and pressure was further reduced to a minimum of 15 mmHg.

Specimens were then assembled on double‐glass sheets with the help of hooks and silicone hoses in vertical shape allowing proper catalysis on both sides simultaneously. This method provided more uniform results and reduced time spent at this stage. Finally, these were placed in a NUV chamber for 72 hours. Near UV‐light was used instead of UV‐light because of the health risk of using UV light chamber in a student access‐free laboratory.

In our experience, better results were obtained by making thin cuts of specimens (<6mm) since it facilitates the visualization of structures and allows the resin to remain fully translucent after being catalyzed.Likewise, results improved when using isopropyl alcohol as a dehydration method because it is less volatile allowing a gradual dehydration to limit dimensional change and shrinkage of the tissue.

Support or Funding Information

Anatomy Laboratory, School of Medicine, Universidad de los Andes.
Potential lung cancer therapy using plant derived cholesterol structural analogs
Authors: Pérez Caraballo Daraishka M. , Torres Anamaris , Milian Melissa , Delgado Yamixa
Published: April 2020

The acquired multidrug resistance syndrome (MDRS) is one of the leading causes for the failure of chemotherapy in lung cancer. Recently plant‐derived triterpenoids (eg, cholesterol‐structural analogs) have showed a wide spectrum of pharmacological activities. In addition, in different studies, anticancer agents have been combined to cholesterol moieties to improve their cellular uptake and target specificity. Herein, we investigated the cytotoxic activity of various triterpenoids cholesterol analogs: oleanolic acid (OleA), ursolic acid (UrA), betulinic acid (BeA), asiatic acid (AsA), lupeol (Lupe), stigmasterol (Stg) and β‐sitosterol (β‐Sito) on non‐small lung adenocarcinoma cells (A549) and normal lung cells (MRC5). From our studies, the IC 50 for most of these cholesterol‐analogs were in the low μM range after 24h of incubation. β‐Sito and Stg did not show any significant cytotoxic pattern in this μM range. In addition BeA and OleA showed the highest therapeutic indexes comparing the cytotoxic effect in A549 vs MRC5 cells. Metabolic activity assays were performed to determine the mechanism of action of these natural compounds. Caspases activity and cell membrane integrity assays were performed and all the cytotoxic triterpenoids induced no activation of caspase‐3, and high membrane permeabilization. Gene expression assays of MDRS‐related genes (EGF, VEGF, Pgp, Bcl‐2, P 53 , NDRG1, GLIPR1) demonstrated the downregulation of some of these genes after the incubation with these triterpenoids. These results demonstrated that even slight structural changes in these cholesterol analogs can influence the cytotoxic response and cellular mechanistic pathways. Complete results will be presented. This study opens promising perspectives for further research on the role of phytochemical triterpenoids, which ultimately will contribute to a more rational application in cancer therapy.

Support or Funding Information

SJB‐Research Center Seed
Hepcidin inhibits ferroportin in the Xenopus oocyte expression system via a calcium‐dependent mechanism that does not involve endocytosis
Authors: Ruwe T. Alex , Qiao Bo , Bonamer John P. , Azucenas Corbin R. , Ganz Tomas , Nemeth Elizabeta , Mackenzie Bryan
Published: April 2020

The cellular iron‐exporter ferroportin (Fpn), under the control of the hormone hepcidin, serves as a key control point in iron homeostasis. The canonical mechanism by which hepcidin suppresses Fpn activity comprises hepcidin binding by Fpn and the subsequent internalization and ubiquitin‐dependent degradation of the transporter. Here we explored hepcidin inhibition of 55Fe efflux from RNA‐injected Xenopus oocytes expressing human Fpn. Hepcidin pretreatment induced a rapid inhibition (t½ = 8.2 ± [SEM] 0.5 min; r2 > 0.99, P < 0.001) of Fpn‐mediated 55Fe efflux. Hepcidin had no effect on Fpn activity when we removed calcium from the pretreatment medium (0 Ca2+, 0.1 mM EGTA). The action of hepcidin was not dependent on endocytosis, a conclusion based on the following observations: (i) Inhibition of Fpn by hepcidin was only modestly temperature‐dependent (Q10= 2.5 ± SEM 1.4 over the range 17–27 °C) whereas endocytosis is expected to be strongly temperature‐dependent, Q10 in the order of 5–17 [Weigel PH and Oka JA (1981) J Biol Chem256, 2615–17]. (ii) Inhibition of Fpn by hepcidin proceeded even in the presence of blockers of clathrin‐mediated (chlorpromazine, Dynasore) and clathrin‐independent (filipin, genistein) endocytosis. (iii) Hepcidin treatment inhibited 55Fe efflux in oocytes expressing the (K→R)8‐Fpn mutant, in which 8 Lys residues required for ubiquitination and endocytosis of Fpn were mutated to Arg, as much as in wildtype Fpn. (iv) Confocal LSM imaging of wildtype Fpn‐GFP and (K→R)8‐Fpn‐GFP expression in oocytes revealed no detectable endocytosis of either protein after treatment with hepcidin. (v) Hepcidin treatment produced no meaningful change in membrane surface area (as determined by capacitance) of oocytes expressing Fpn, despite reducing Fpn‐mediated iron transport by 65%. Our data reveal that hepcidin can directly block Fpn activity via a Ca2+‐dependent mechanism that does not involve endocytosis. We speculate that the Xenopus oocyte lacks the specific signal by which hepcidin triggers Fpn internalization. The Xenopus oocyte is therefore an ideal model system in which to study the mechanism by which hepcidin directly blocks Fpn activity.

Support or Funding Information

NIH grants R01 DK107309, P30 DK078392, and R25 HL115473, and the American Physiological Society
Intrinsic Versus Extrinsic Intra‐Subject Variability in Sweat Rate of Individual Eccrine Glands
Authors: Moore Jeff , Woolf Emily , Wells Nicole , Northway Sarah , Buono Michael
Published: April 2020

The purpose of the current study was twofold. First, it was to determine the intra‐subject variability of sweat rate per gland for a given skin location during exercise in the heat. Second, it was to determine the relative importance of intrinsic vs. extrinsic factors as the source of the intra‐subject variability in sweat rate per gland. Sweat rate of individual eccrine glands on the forearm was measured following pilocarpine iontophoresis and during exercise in the heat. In five subjects during exercise in the heat the measured sweat rate for individual forearm eccrine glands (n= 500) ranged from 0.5 nL/gland/min to 16 nL/gland/min, or over a 30‐fold difference. The mean ± SD intra‐subject coefficient of variation in sweat rate per gland was 36±5% and 49±10% (p = 0.008) following pilocarpine iontophoresis and during exercise in the heat, respectively. Such results suggest that intrinsic factors (i.e., sweat gland size and cholinergic sensitivity) contribute approximately three times more than extrinsic factors (i.e., sweat gland nerve fiber density and threshold amplitude) towards explaining the large intra‐subject variability in sweat rate per gland seen during exercise in the heat.
SPARC: Determining the Projections and Expression Profiles of Neurons of the Stomach‐Brain Axis in Rat
Authors: Furness John B. , Hunne Billie B. , Di Natale Madeleine B. , Molero Juan C. , Stebbing Martin C.
Published: April 2020

Effectors in the stomach receive motor innervation from intrinsic (enteric) neurons, nuclei in the brain stem, the spinal cord and other gut regions. Sensory signals are conducted to the brain, spinal cord and sympathetic ganglia. We are analyzing the circuits by selective pathway lesions, orthograde and retrograde tracing, RNAScope and immunohistochemistry. Regions of stomach were denervated by myectomy (removing a region of myenteric ganglia), bilateral vagotomy or sympathectomy, or combined surgeries. Retrogradely transported fluorescent microbeads were injected selectively into the gastric mucosa or muscle layers. Neuronal markers were localized by multi‐labeling immunohistochemistry. When a region of longitudinal muscle and myenteric ganglia was removed, and 7–10 days allowed for fiber degeneration, nerve fibers in the circular muscle and muscularis mucosae were no longer detected. Two major nerve fiber types innervated the muscle, fibers immunoreactive for neuronal NOS and VIP that are deduced to be inhibitory motor neurons and fibers that were labelled by tachykinins and cholinergic markers that are deduced to be excitatory motor neurons. Some inhibitory fibers were also immunoreactive for gastrin releasing peptide (GRP). The muscle was also innervated by sympathetic (tyrosine hydroxylase positive) nerve fibers. The mucosa received a mixed motor and sensory supply from myenteric ganglia, submucosal ganglia, the vagus, and sympathetic pathways. Denervation studies showed that VIP fibers arose from both myenteric ganglia and the nodose ganglia; thus VIP is in subpopulations of motor and of sensory fibers. Some VIP fibers ended in close relation to ghrelin‐containing enteroendocrine cells. There were few sympathetic endings in the mucosa and CGRP fibers were also rare. Arteries in the gastric wall were triply innervated by sympathetic noradrenaline/ NPY fibers, tachykinin fibers and VIP fibers. The origins of the perivascular VIP fibers have yet to be determined. Injection of retrograde tracer into the gastric mucosa labeled large populations of nodose ganglion neurons. Some nodose ganglion neurons were labeled both from the gastric mucosa and from the external muscle. The chemical phenotypes of myenteric and submucosal ganglia are being investigated. VIP‐immunoreactive cell bodies were numerous in submucosal ganglia. We conclude that there is a complex interplay between nerve endings originating in myenteric, submucosal, nodose, sympathetic and spinal ganglia in gastric effector tissues.

Support or Funding Information

NIH/SPARC
Cinaciguat Reduces Prolyl Hydroxylase 2 (PHD2) Protein Expression in Chronically Hypoxic and Pulmonary Hypertensive Newborn Lambs
Authors: Beñaldo Felipe Augusto , Araya-Quijada Claudio , Serón-Ferre María , Herrera Emilio , Ebensperger Germán , Reyes Roberto , Llanos Aníbal
Published: April 2020

Newborn lambs at high altitudes (3,600 m) develop pulmonary hypertension due to a sustained hypoxic vasoconstriction followed by a pathological remodeling of small pulmonary arteries (SPA). The latter is produced by several agents, including the transcription factor Hypoxia Inducible Factor‐1α (HIF‐1α). HIF‐1α increases the pulmonary vascular remodeling in chronic hypoxia and the enzyme PHD‐2 is a key factor participating in the degradation of HIF‐1α. Cinaciguat an activator of sGC is able to decrease HIF‐1α, in adults, and increase HO‐1 in neonatal lambs. This enzyme reduces reactive oxygen species (ROS) and cytokines, whereas no information in PHD‐2 and cinaciguat is available. Therefore, we use two groups of 6 newborn lambs each, that were catheterized under general anesthesia and a Swan Ganz catheter was placed into the pulmonary artery, to measure pulmonary arterial pressure (mPAP), cardiac output (CO) and calculate pulmonary vascular resistance (PVR). The groups received, either the activator of sGC (cinaciguat 35 μg kg−1) or vehicle (DMSO : NaCl 0,9%, 1:10), for seven days. Additionally, we took lung samples to perform histology (muscle & adventitia layers in small pulmonary arteries, SPA) and western blots (HIF‐1α and PHD‐2). Forty‐eight hour after the end of treatment, euthanasia was accomplished. All procedures were approved by the local Bioethical Committee (CBA#0643 FMUCH). Cinaciguat did not change the mPAP but did vary the PVR. Mean PAP was not modified because CO increased. Concordant with the lower PVR there was a reduction of the muscular and adventitia layers in SPA. Further, cinaciguat did not modify the HIF‐1α protein expression but showed a decrease in PHD‐2 in pulmonary hypertensive newborn lambs. If ROS and cytokines are diminished (unmeasured) by cinaciguat, both factors could generate a reduction in HIF‐1α, that could lessen PHD‐2 (feedforward). The latter should produce a decrease in HIF‐1α hydroxylation and therefore a reduced destruction in the proteasome, which would lead to an increase in HIF‐1α availability, and eventually to its initial state (feedback). HIF‐1α did not decrease because has a short half‐life in contrast with PHD‐2 that has a half‐life over 48h. In conclusion, the decrease in PHD‐2 could be the consequence of an initial HIF‐1α reduction, producing a diminution in muscle and adventitia layers in small pulmonary arteries consistent with a lesser PVR. The reduction of HIF‐1α could be considered a useful tool to treat the pulmonary hypertension of the neonate.

Support or Funding Information

Conicyt 21191677, Fondecyt 1140647, Chile.
The Effect of Creatine Monohydrate Supplementation on Tissue Creatine Concentrations in Male and Female Rats
Authors: Dunham Tyler , Murphy Jensen , Colonna Kaitlyn , Fajardo Val , MacPherson Rebecca , Ward Wendy , Roy Brian
Published: April 2020

Background

Creatine is an essential compound that can store chemical energy and act as an energy buffer during times of metabolic stress in mammals. Creatine can be synthesized endogenously, however dietary supplementation with creatine monohydrate (CM) is a common medical intervention and is used as a common ergogenic aid in athletes. The aim of this study was to investigate the effect of increasing doses of dietary creatine on creatine uptake in brain, heart, skeletal muscle, and liver tissues in male and female rats in preparation for future dietary interventions.

Methods

16 male (536g—670g) and 16 female (339g—487g) 12 week old Sprague‐Dawley rats were given access to drinking water with 0 g/L, 2.5 g/L, 5 g/L, or 10 g/L CM and 1% sucrose for 8 weeks. Free creatine and phosphocreatine tissue concentrations were measured using fluorometric assays and combined to report total creatine compound (TCr) concentrations in brain (BR), heart (HRT), liver (LIV), and skeletal muscle [soleus (SOL), extensor digitorum longus (EDL), and gastrocnemius (GAS)] tissues after the 8‐week dietary intervention.

Results

Males were significantly heavier than females (p<0.001) but there were no differences in body weight between groups of either sex. TCr concentrations in BR were higher with 2.5 g/L (p<0.01), 5 g/L (p<0.05), and 10 g/L (p<0.001) CM compared to control, with only the 2.5 g/L CM dose resulting in a higher TCr concentration in females (p<0.05) compared to males. TCr concentrations in SOL were higher with 5 g/L (p< 0.01) and 10 g/L (p<0.001) CM compared to controls. In EDL and GAS TCr concentrations were higher compared to controls with 2.5 g/L (p<0.01, p<0.05), 5 g/L (p<0.001, p<0.05), and 10 g/L (p<0.001, p<0.01) CM. TCr in GAS showed a trend with females (p<0.10) being higher than males. TCr concentrations in HRT of females were higher with 10 g/L (p<0.01) CM compared to controls and trending towards significance with 5 g/L (p<0.10) CM as compared to controls. TCr concentrations in HRT with 10 g/L CM were significantly higher in females (p<0.001) compared to males. No differences in TCr were observed between groups in LIV.

Conclusions

CM supplementation provided in water with 1% sucrose for 8 weeks leads to an increase in TCr in brain, heart, and skeletal muscle, but not in liver. Females appeared to be more responsive to CM supplementation in HRT, especially at the highest dose suggesting that dietary interventions involving CM may affect males and females differently.

Support or Funding Information

Supported by NSERC, Canada.
Unraveling the Different Network States of the Respiratory Network Using Experimental and Computational Approaches.
Authors: Bush Nicholas Edward , Baertsch Nathan , Burgraff Nicholas , Harris Kameron , Ramirez Jan-Marino
Published: April 2020

Rhythm generation in the respiratory system must be sufficiently robust to ensure continued rhythmic activity in a variety of challenges, but flexible enough to meet the various physiological demands of the organism. The control of frequency and regularity of the respiratory rhythm critically depends on the interplay between inhibitory and excitatory synaptic activity and intrinsic membrane properties in the pre‐Bötzinger complex network. Optogenetic manipulation allows for selective control of these inhibitory and excitatory components. We find that increasing inhibitory drive during the inspiratory phase drives increases in burst frequency, whereas increasing inhibition in the inter‐burst interval decreases frequency. This frequency effect critically depends on the refractory period that is controlled by the network excitability during the inspiratory burst. The frequency is also differentially controlled by aminergic and peptidergic neuromodulators. Our experimental data indicate that Substance P increases the respiratory frequency by acting specifically on the pre‐inspiratory period. The frequency effects of opioids on the other hand depend on excitatory synaptic mechanisms and the persistent sodium current (INaP). Weakening the INaP current pharmacologically biases the network towards glutamatergic mechanisms, which increases the opioid sensitivity, while strengthening the INaP current pharmacologically or by exposing the network to hypoxia decreases the opioid sensitivity. To complement our experimental investigations of rhythm generation and neuromodulation in the respiratory network, we implement our published computational model (Harris et al. 2017, Journal of Neurophysiology). This model incorporates cell type specific and temporally constrained currents that can be used to mimic experimental manipulations such as optogenetics. This approach will provide an in silico tool to dissect and predict network responses to such manipulations.

Support or Funding Information

P01 HL090554; R01 HL144801; R01 HL126523
Potent and selective inhibition of anion exchange activity of SLC26A3 by DI330
Authors: Park Jinhong , Namkung Wan
Published: April 2020

SLC26A3 (downregulated in adenoma, DRA), Cl−/ anion exchanger, is highly expressed in the luminal membrane of intestinal epithelial cells, and loss‐of‐function mutations in this gene result in congenital chloride diarrhea caused by detect of chloride absorption and bicarbonate secretion in the intestine. Here, we report development of the selective small‐molecule inhibitors of DRA for the investigation of the effect of DRA inhibition on constipation and metabolic acidosis. A screen of ~50,000 compounds using a cell‐based high‐throughput screening (HTS) assay revealed four novel chemical compounds of DRA inhibitors that fully blocked Cl−/I− exchange activity of DRA with IC50 < 10 μM. The hit compounds strongly inhibited both Cl−/I− and Cl−/HCO3− exchange activity of DRA. Structure‐activity relationship were performed on the potent inhibitor classes identified in the screening. The potent inhibitor, DI330, significantly blocked Cl−/I− exchange activity of DRA (IC50 of ~30 nM) but it did not affect the activity of SLC26A4 (pendrin), SLC26A7, SLC26A9, CFTR and ANO1. The DI330 identified here may be useful tools for pharmacological dissection of DRA and drug discovery for treating constipation and metabolic acidosis.
Neurovascular Coupling is Not Attenuated During Reflex‐Mediated Sympathetic Activation via Lower Body Negative Pressure in Humans
Authors: Samora Milena , Vianna Lauro C. , Carmo Jake C. , Macedo Victor , Dawes Mathew , Phillips Aaron A. , Paton Julian F.R. , Fisher James P.
Published: April 2020

Introduction

Cerebral blood flow is tightly coupled with local neuronal activation and metabolism (i.e., neurovascular coupling; NVC). Attenuated NVC has been identified in several chronic diseases, including hypertension, but the underlying mechanism(s) remains unknown. Raised peripheral sympathetic vasoconstrictor activity can play a malevolent role in the pathology of hypertension and might also contribute to the blunted NVC as cerebral arteries are autonomic innervated. However, the role of sympathetic nervous system in the regulation of cerebral blood flow remains controversial. Given this, the purpose of the current study was to test the hypothesis that reflex‐mediated sympathetic activation via lower body negative pressure (LBNP) blunts NVC in humans.

Methods

NVC was assessed using a visual stimulation protocol (5 cycles of 30 s eyes closed and 30 s of reading) in 10 young and healthy participants (3 women; aged 25 ± 3 years). In a randomized and counterbalanced order, NVC assessments were made under control conditions and during graded sympathetic activation via LBNP at −20 mmHg and −40 mmHg. Posterior (PCA) and middle cerebral artery (MCA) mean blood velocity (vmean) were determined (transcranial Doppler ultrasound). Vertebral artery blood flow (VAflow) was measured using duplex Doppler ultrasound and vessel wall tracking software. Mean arterial pressure (finger photoplethysmography), stroke volume (Modelflow), heart rate (electrocardiogram) and the partial pressure of end‐tidal carbon dioxide (PetCO2; capnograph) were obtained. Data are presented as mean ± SD.

Results

LBNP to −20 mmHg and −40 mmHg evoked an anticipated reduction in stroke volume and increase in heart rate, while mean arterial pressure was unchanged. Prior to visual stimulation, PCAvmean and MCAvmean were not different between control, −20 LBNP and −40 LBNP conditions (P>0.05), while VAflow was reduced at −40 mmHg (P=0.02 vs. control). Under control conditions, the NVC test evoked a robust increase in PCAvmean (Δ22 ± 4%), a modest increase in MCAvmean (Δ10 ± 3%), and a moderate rise in VAflow (Δ14 ± 5%). The magnitude of these responses was unaffected by LBNP at both −20 mmHg and −40mmHg (PCAvmean Δ23 ± 4% and Δ23 ± 4%; MCAvmean Δ10 ± 1% and Δ11 ± 4%; VAflow Δ15 ± 3% and Δ16 ± 6%; P>0.05). PetCO2 decreased at −20 mmHg (37.0 ± 3.1 mmHg) and −40 mmHg (36.3 ± 3.1 mmHg) LBNP trials compared to control (40.2 ± 3.7mmHg; P=0.004).

Conclusion

In summary, these findings indicate that reflex‐mediated sympathetic activation via LBNP does not blunt the NVC response in healthy humans.

Support or Funding Information

Funding received from Coordination for the Improvement of Higher Education Personnel (Finance Code 001) (MS), the University of Auckland (JPF, JFRP) and the Health Research Council of New Zealand (JPF, JFRP).
Role for Asef in the Maturation and Maintenance of Endothelial Cell‐Cell Adhesions
Authors: Kitt-Hopper Khameeka
Published: April 2020

The formation of cell‐cell adhesions involves the spatiotemporal regulation of protein complexes, including cadherins and reorganization of the actin cytoskeleton by Rho GTPases. Previous work identified a cooperative role between cadherins and nectins to induce and rapidly suppress Rac1 GTPase activity during de novocell adhesion in epithelial cells. In this study, the role of Asef (guanine nucleotide exchange factor (GEF) for Rac1) on the assembly and maturation of cell‐cell contacts in human umbilical vein endothelial cell (HUVEC) is examined. The Rho GTPases, Rac1 and RhoA, have been previously shown to regulate endothelial cell permeability, but the proteins involved in controlling the activity of these Rho GTPases during endothelial junction assembly and maintenance remain relatively unknown. The current study shows that endogenous Asef is gradually recruited to cell‐cell contacts 3–5 hours after initiation of cell‐cell adhesion, and localizes to established endothelial cell‐cell contacts. Knockdown of Asef reduces the amount of VE‐cadherin and Asef’s only known binding partner, Adenomatous Polyposis Coli (APC) at cell‐cell contacts. Significantly, knockdown of Asef causes an increase in paracellular permeability of HUVEC monolayers and a decrease in HUVEC cell adhesiveness. These data indicate a role for Asef in maintaining the integrity of endothelial cell‐cell adhesions, and potentially a specific role at tight junctions. Future work aims to decipher whether Asef regulates the recruitment and maintenance of adherens junctions, tight junctions or both at cell‐cell contacts, and the signaling pathways involved in the spatiotemporal regulation of these protein complexes at cell‐cell adhesions in various adherent cell lines.
Exogenous lactate increases fat oxidation in resting rats
Authors: Yoo Choongsung , Kim Jisu , Kyun Sunghwan , Kreider Richard , Lim Kiwon
Published: April 2020

Background

Lactate had been traditionally considered a fatigue‐associated byproduct of high intensity exercise. Although several physiological roles of lactate have been revealed in the last decades, its effects on energy metabolism and substrate oxidation remain unknown. Therefore, here, we investigated the effects of lactate on the energy metabolism of resting rats.

Methods

Male rats were divided into control (Con; distilled water), caffeine (Caf; 10 mg/kg), lactate (Lac; 4 g/kg), and caffeine plus lactate (Caf+Lac; 10 mg + 4 g/kg) groups. After oral administration of treatments, animals were evaluated for resting energy expenditure, biochemical blood parameters, and mRNA levels of key genes involved in energy metabolism in the soleus muscle.

Results

Groups did not differ in total energy expenditure throughout the 6‐h post‐treatment evaluation. Within the first 4 h, the Lac and Caf+Lac groups showed higher fat oxidation rates than that of the Con group. Carbohydrate oxidation rates were similar between Lac and Con groups. Lactate treatment decreased blood free fatty‐acid levels and increased the mRNA expression of fatty acid translocase (FAT/CD36) and peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α) in skeletal muscle.

Conclusions

Our findings indicate that lactate treatment can enhance fat metabolism in whole body and skeletal muscle.

Support or Funding Information

This paper was supported by the KU Research Professor Program of Konkuk University.
Biochemical analysis of PriA Helicases from Gram‐positive Bacteria involved in DNA Replication Restart
Authors: Hsiao Chwan-Deng , Lin Min-Guan , Li Yi-Ching , Sun Yuh-Ju
Published: April 2020

DNA replication forks often encounter template DNA lesions, which can stall progression of the replication forks. In gram‐positive bacteria, the PriA‐dependent pathway is the major replication restart mechanism and it requires several primosome proteins. Among them, the PriA protein—a 3′ to 5′ superfamily‐2 DNA helicase—is the key factor recognizing DNA lesions and it further recruits other proteins. Here, we investigated the PriA ATPase and helicase activities of Streptococcus pneumoniae (SpPriA) and Geobacillus stearothermophilus (GstPriA) through biochemical and kinetic analyses. Using various DNA duplexes with unstructured single‐stranded regions, we observed distinct unwinding abilities for GstPriA and SpPriA according to increased GC content of duplex DNA substrate. In contrast to the unwinding activity of GstPriA helicase, we found that SpPriA is unable to unwind duplex DNA substrate with high GC content. However, the presence of DnaD loader protein allows SpPriA to unwind such duplex DNA. Our findings suggest that SpPriA possesses limited unwinding ability in high GC DNA and that DnaD acts as an accessory protein to destabilize stalled replication forks through its single‐stranded DNA binding ability, thereby enhancing PriA unwinding activity.
Lipid metabolism changes in C. elegans models of aging
Authors: Chan Jason P. , Radeny Joycelyn , Wang Lu
Published: April 2020

Lipid metabolism plays major roles in cellular membrane structure and fluidity, signaling, and energy storage. Lipids have a tremendously diverse composition, structures, and saturation levels, making their cellular function complex. Recent data have suggested that presence of particular lipids correlate with aging physiology, including cardiovascular and neurodegenerative diseases. Furthermore, genetic or pharmacological manipulation of lipid enzyme function impacts aging, including lifespan and healthspan (period of life in good health). Using genetic models of aging in Caenorhabditis elegans, we performed a lipidomics study examining a panel of lipids, including free fatty acids, triacylglycerols, sphingomyelins, and other phospholipids. We measured 888 lipids, and 702 lipids were present in 80% of our samples. We tracked these lipids in 1, 5, and 10 day old adult worms, and analyzed patterns across age and mutant background. In particular, we highlight findings in a sphingolipid metabolism mutant, asm‐3 (acid sphingomyelinase), which has an increased lifespan. We compare these to physiological responses of asm‐3 mutants in response to stress. Together, we hope this analysis adds to our understanding of the biology of aging and yields novel lipid targets that may impact lifespan and healthspan.

Support or Funding Information

NIH 2R15AG052933 and NIH 1S10OD021562
Increasing Success of Underserved Students in Human Anatomy
Authors: Akacem Lameese Dana , Ciancanelli Rebecca
Published: April 2020

The Student Academic Success Center is a multicultural learning community that supports students who are low income, first generation, and underrepresented at the University of Colorado Boulder. In order to improve undergraduate science outcomes for students who are traditionally underserved in higher education, our department offers small sections of high risk gateway courses. Prompted by low performance in the large main campus section of Human Anatomy, we developed a small section this course for our department. Our small section of Human Anatomy used the same lecture material and exams as the main campus sections. We added a recitation that met for 75 minutes per week. During the recitation, students completed a variety of activities including concept maps, worksheets, and clay‐building facilitated by undergraduate Instructional Assistants. Students also completed weekly quizzes during the recitation. Students in our community who took the main campus section of Human Anatomy had an average success rate (defined as a final grade of A or B) of 40% and a D/F rate of 31%. In the small section of Human Anatomy, we observed an average success rate of 45% and D/F rate of 7%. These results indicate that the small class model embedded in a community with extra support may increase the success of underrepresented students in Human Anatomy and has implications for increasing persistence of a diverse student body in health career trajectories.
Structural‐Based Drug Discovery Targeting PCNA: A Novel Cancer Therapeutic
Authors: Jossart Jennifer , Kenjic Nikola , Quninoez Robert , Perry Jeff
Published: April 2020

Proliferating Cell Nuclear Antigen (PCNA), which is a member of the sliding clamp family, critically functions as a hub for proteins that are involved in DNA metabolism events, by bringing them into close proximity with DNA. A unique isoform of PCNA has been defined as playing a critical pathological role in multiple tumors, including breast cancer and neuroblastoma. Notably, this cancer associated isoform of PCNA (caPCNA) is highly expressed in cancer cells, but not at significant levels in non‐malignant cells. Thus, we hypothesize caPCNA could act as a novel target for the development of cancer‐selective drugs. By using computer‐aided drug design (CADD) driven approaches, our collaborators at the City of Hope Medical Center have recently identified first‐in‐class small molecules capable of targeting caPCNA and killing caPCNA‐driven cancer cells. Our aim is to characterize these novel compounds by protein crystallography, to aid their development into more drug‐like molecules. Thus far, we have discovered a novel apo‐PCNA crystallization condition diffracting to 2.1 Å that can be potentially used in our analyses. We have also begun fragment‐based drug discovery (FBDD) studies, to identify novel chemical groups that could be utilized in our drug‐discovery efforts. Through thermal shift screening assays, we have revealed over 70 potential fragment binders that are undergoing structural characterizations. Thus, through combined CADD, FBDD, biochemical and cell‐based assays, our analyses of initial CADD‐based compounds and fragment hits can be used to develop compounds suitable for preclinical studies, and ideally, open a novel therapeutic avenue for the selective targeting of cancers.

Support or Funding Information

DOD CDRMP BCRP Level 2 award‐ “Breast Cancer Selective Therapeutic Development: Lead Optimization of a Small Molecule Inhibitor of The Breast Cancer Selective Hub Protein caPCNA”
GDNF drives collecting duct‐like tubuloids in novel 3D in vitro system
Authors: Dixon Eryn E. , Watnick Terry , Welling Paul A. , Woodward Owen M.
Published: April 2020

Three‐dimensional (3D) in vitro model systems have provided critical insights into the function and structure of many tissues, including lung, intestine, and brain. In recent years, the development of kidney organoids has been rapidly progressing with intricate differentiation and characterization of embryonic pluripotent and adult stem cells. These systems have significant drawbacks, including nascent phenotypes and complex differentiation of many cell types. Here, we have focused on understanding the specific cues in culture necessary to isolate nephron segments and developed a novel 3D in vitro system that employs mouse kidney tubule fragments containing endogenous progenitor cells to yield a collecting duct‐like (Dolichos biflorus agglutinin, DBA+) phenotype. After initial seeding of tubule fragments from postnatal mouse kidneys in Matrigel, a subset of healthy cells displayed strong expression of the renal stem cell and mesenchymal mesoderm (MM) marker, Six2. Six2 progenitor cells during kidney development secrete the growth factor glial derived neurotrophic factor (GDNF), stimulating the elongation and branching of the ureteric bud (UB), eventually becoming the collecting duct system. We harnessed the presence of the Six2 progenitor cells and used a 24 hour pulse of exogenous GDNF to aid in the promotion of UB like cells to promote the growth of collecting duct‐like structures. The GDNF pulse produced a significant increase in the expression of Gata3 and Wnt11 in the resulting tubuloids, without altering the amount of Six2 expression, suggesting preferential growth of collecting duct tubuloids. The addition of a GDNF pulse was augmented with hepatocyte (HGF), epidermal (EGF), and fibroblast (FGF) growth factors, to increase the branching of the growing collecting duct‐like tubuloids, resulting in more differentiated, ciliated structures (E‐cadherin+, ZO‐1+, Na+/K+‐ATPase+, α‐tubulin+) and significantly more tubules as a percentage of total structures. Expanding the applications of this system and using a Pkd2fl/fl Pax8 rtTA TetOCre +mTmG mouse model, we have applied these culture principles to autosomal dominant polycystic kidney disease, where differentiated epithelial structures can be tracked for morphological and organizational changes before and after inactivation of Pkd2 by addition of doxycycline. Therefore, the utility of this new collecting duct‐like culture model is broad and dynamic, providing more insight into culture manipulation of primary mouse cells, in vitro differentiation, and new physiological applications.

Support or Funding Information

PKD Foundation, NIDDK (F31 DK117579), and Baltimore PKD Research and Clinical Core Center (P30 DK090868)
Diversity of nasal and intestinal microbiome in the first 24 hours of life in newborns
Authors: Carbajal Patricia Bautista , Pérez-Gopar Martha A. , Espinoza-Torres Bogart , Hernández-Pérez Brenda , Rodríguez-Bernabe Alma A. , García-León Miguel L. , RiveraÁvalos Carlos E. , Santaella-Servin Monserrat , López-soto Iris I. , Ángel-Ambrocio Antonio H. , Wong-Chew Rosa M.
Published: April 2020

Introduction

Disbyosis in the intestinal microbiome has been associated to disease. It has been described that newborns are sterile and get colonized in the first days. Little is known about the respiratory microbiome in the first hours of life. The aim of the study was to compare the nasal and intestinal microbiome in newborns by c‐section (C) and vaginal delivery (V).

Methods

Women who gave birth in the previous 24 hours were invited to have their babies to participate in the study. Nasal swabs and feces were obtained. DNA extraction from nasal and fecal samples was performed, V3‐V4 of the RNA 16S ribosomal subunit was determined by PCR and NG sequencing of this region was done for taxonomic identification of the microbiome.

Results

30 C newborns and 30 V were included. There was a 10% detection of V3‐V4 in nasal washes in C compared to 50% in V, and 46% vs 53% detection of V3‐V4 in feces of C and V, respectively. Phylum analysis showed C vs V: Firmicutes in 50% vs 22% Proteobacteria 39% vs 75% Actinobacteria 6% vs 2%, Bacteroidetes 4% vs 0.9%, Verrucomicrobia 0.8% vs 0% and not classified 0.6% vs 0.2%, respectively. In nasal samples C vs V we found: Firmicutes 49% vs 45%, Proteobacteria 36% vs 27%, Actinobacteria 4.4% vs 11%, Bacteroidetes 7.6% vs 10%, Fusobacteria 0.2 vs 3% and not classified 2.8% vs 4%, respectively.

The genus analysis showed in C newborns feces: Enterococcus in 20%, Streptococcus 2%, Lactobacillae 1%, Staphylococcus 22%, Clostridiales 2%, Escherichia‐Shigella 22%, Enterobacteriacea 13%, Acinetobacter 1%, Bifidobacterium 4%, Actinomycetales 2%, Bacteroides 2%; in V newborns Enterobacteriacea in 27%, Escherichia Shigella 47%, Clostridiales 18%, Sarcina 1%, Leuconostoc 1%, Bifidobacteria 2%. In nasal samples of V newborns there was a higher genus diversity (30) than C newborns (22) and less Stahylococcus (4% vs 14%) and Streptococcus (11% vs 32%), respectively.

Conclusions

A lower proportion of C newborns are colonized in the respiratory airway in the first hours of life. C newborns have Staphylococus and Streptococcus in their intestinal microbiome which can be found in the skin of their mothers while in V newborns Enterobacteria is predominant. A higher genus diversity and less Streptococcus and Staphylococcus is observed in the nasal microbiome of V newborns compared to C newborns.

Support or Funding Information

This study was funded by the Consejo Nacional de Ciencia y Tecnología (CONACYT) Mexico, grant number SALUD 2015‐878, PI Rosa M. Wong‐Chew.