bluuebunny/crossref_metadata_2025 huggingface dataset:
Aversive Effects of the Kappa Opioid Agonist U‐50488 Alone and in Combination with the Cannabinoid Agonist CP 55,940 in Male Rats
Authors: Friend Ashton Jeremy , Erwin Laura Lynn , Aslan Abdurrahman Jeremy , Smith William L. , Nilges Mark R. , Winsauer Peter J.
Published: April 2020

Kappa (κ) opioid receptor agonists can produce prominent analgesic and diuretic effects when administered alone and these effects can be enhanced by the co‐administration of cannabinoids such as CP 55,940. The purpose of the present experiment was to determine if cannabinoids can also enhance the aversive effects of kappa agonists, because these effects substantially reduce their clinical utility as analgesics and aquaretics. To study the aversive effects of kappa agonists and their interaction with cannabinoids, nine male Long‐Evans rats were trained to respond for food under a fixed‐ratio (FR) schedule of food presentation and then implanted with intravenous (i.v.) catheters. Following recovery, the subjects were transitioned to a multiple FR, FR schedule. More specifically, in the presence of a single white stimulus light, food was presented under a fixed‐ratio 30 (FR‐30) schedule (non‐infusion component), whereas presence of both this white stimulus light and a green stimulus light, an i.v. infusion was presented on the first response of each FR and food was presented on the thirtieth FR response (infusion component). When responding was consistent in both components, varying infusion doses of U‐50488 (0.032 – 0.18 mg/kg) replaced the baseline saline infusion until one of three criterion was met: 3 days in which the variability of responding did not vary by ± 20% of the mean, 3 days of responding at less than 0.3 responses per sec, or a maximum of 8 days. After the criterion was met for each dose substitution, the subjects returned to the baseline in which saline served as the infusion and responding was similar between components. To study the interaction of CP 55,940 with the effects of U‐50488, single acute injections of a dose of CP 55,940 (0.056 mg/kg) were administered prior to sessions in which a dose of U‐50488 was available in the infusion component. Each dose‐infusion combination was administered until subjects met the criterion. These effects were then compared to sessions in which one of two doses of naltrexone (1, 3.2 mg/kg) was administered prior the same U‐50488 infusion doses. When infusions of U‐50488 replaced saline, low infusion doses (e.g., 0.056 mg/kg/infusion) selectively suppressed responding in the infusion component, while higher infusion doses (e.g., 0.18 mg/kg/infusion) decreased responding in both components as the total dose of U‐50488 for the session increased. Administration of CP 55,940 did not produce a marked enhancement of the suppressive effects of U‐50488 at the dose tested, and surprisingly, neither dose of naltrexone antagonized its suppressive effects. Together, these data demonstrate the aversive effects of U‐50488 alone, the absence of a marked enhancement by at least one cannabinoid agonist, and the surprising absence of antagonism by the opioid receptor antagonist naltrexone.
Direct reprogramming of dermal fibroblasts derived from Northern elephant seals into muscle cells
Authors: Lam Emily K. , Torres-Velarde Julia Maria , Allen Kaitlin , Crocker Daniel , Vazquez-Medina Jose Pablo
Published: April 2020

Ex vivo tissue culture is a powerful technique that allows the study of complex cellular mechanisms that are relevant to physiological responses in animals while overcoming the challenges presented by studying animals that are not tractable. In a primary cell culture system, certain proliferating cells can be functionally reprogrammed into other cell types via overexpression of key genes. Dermal fibroblasts can be reprogrammed into muscle progenitor cells (myoblasts), which are often challenging to obtain but offer a unique system to study metabolic responses, by overexpression of the myogenic transcription factor myod. We isolated dermal fibroblasts from Northern elephant seal (NES) skin samples and propagated them in primary culture. NES skin fibroblasts stained positive for the Platelet‐derived growth factor receptor. Primary NES fibroblasts were amenable to eGFP transfection by both, lipofection and electroporation. Maximal transfection efficiency was achieved using the Neon Electroporation system and corresponded to 55% of living cells. We overexpressed myod in NES fibroblasts and conducted antibiotic selection with 2 ug/mL puromycin for 3 days. As expected, expression of myod was significantly higher in transfected cells according to qPCR analysis (t‐test p< 0.05). Treatment with small molecules (CHIR99021, Forskolin and Repsox) enhanced myod expression. Furthermore, fibroblasts overexpressing myod expressed downstream markers of myogenesis (myogenin, myosin heavy chain 1 and myosin heavy chain 8) and the effect was enhanced when myod‐overexpressing cells were supplemented with small molecules. We are currently evaluating the capacity of myod‐overexpressing dermal fibroblasts to differentiate into myotubes and comparing differentiation and metabolic profiles with primary NES myoblasts. Establishing differentiated muscle fibers from other mature cell types such as fibroblasts could provide a unique platform to conduct mechanistic studies in species where muscle tissue samples cannot be obtained from live animals.
The Effects of Bcl6 KO in Mouse Model of Hearing
Authors: Morrill Seth , He David
Published: April 2020

Although inner and outer hair cells (IHCs and OHCs) in the inner ear are both vulnerable to noise and ototoxic drugs, IHCs are less susceptible to those insults. The molecular mechanisms of such differential vulnerability are unknown. Hair cell‐specific transcriptome analysis shows that Bcl6, a known anti‐apoptotic gene, is differentially expressed in IHCs compared to OHC with a 3‐fold difference. We hypothesize that higher expression of Bcl6 in IHCs contributes to relative resilience of IHCs to aging, ototoxic and mechanical assault. Here, we examine the role of Bcl6 on survival and differential vulnerability of IHCs and OHCs using a Bcl6 conditional knockout mouse model generated by mating the B6.Cg‐Tg(Atoh1‐cre)1Bfri/J mouse line with the B6.129S(FVB)‐Bcl6tm1.1Dent/J from Jackson Labs. Auditory Brainstem Response (ABR)‐based thresholds were determined using tone pips with frequencies between 4–50 kHz in homozygous and wild type littermate mice at postnatal ages of 30 and 90 days. Separately, application of mechanical insult through high intensity sound was used to determine if mechanical insult is sufficient to make IHCs and OHCs equally vulnerable to apoptosis. Hair cell survival and morphology from the three cochlear locations (representing apical, middle and basal turns of the cochlea) were examined using confocal microscopy. A diminished hearing threshold and loss of hair cells suggests that Bcl6 is necessary for hair cell maintenance and survival. Two lines of work are currently under way: 1) assessing changes in gene expression (transcriptomes) and identification of downstream targets of Bcl6 in hair cells using hair cells isolated and collected from three genotypes, and 2) applying ototoxic insult to mice to determine whether loss of function due to Bcl6 deletion would make IHCs equally vulnerable to noise and ototoxic insults.
‐ Two Alternative Mechanisms Regulate the Onset of Chaperone‐mediated Assembly of the Proteasomal ATPases
Authors: Park Soyeon , Nahar Asrafun
Published: April 2020

The proteasome holoenzyme is a molecular machine that degrades most proteins in eukaryotes. In the holoenzyme, its heterohexameric ATPase injects protein substrates into the proteolytic core particle, where degradation occurs. The heterohexameric ATPase, referred to as ‘Rpt ring’, assembles through six ATPase subunits (Rpt1‐Rpt6) individually binding to specific chaperones (Rpn14, Nas6, Nas2 and Hsm3). Here, our findings suggest that the onset of Rpt ring assembly can be regulated by two alternative mechanisms. Excess Rpt subunits relative to their chaperones are sequestered into multiple puncta specifically during early‐stage Rpt ring assembly. Sequestration occurs during stressed conditions, for example heat, which transcriptionally induce Rpt subunits. When the free Rpt pool is limited experimentally, Rpt subunits are competent for proteasome assembly even without their cognate chaperones. These data suggest that sequestration may regulate amounts of individual Rpt subunits relative to their chaperones, allowing for proper onset of Rpt ring assembly. Indeed, Rpt subunits in the puncta can later resume their assembly into the proteasome. Intriguingly, when proteasome assembly resumes in stressed cells or is ongoing in unstressed cells, excess Rpt subunits are recognized by an alternative mechanism—degradation by the proteasome holoenzyme itself. Rpt subunits undergo proteasome assembly until the holoenzyme complex is generated at a sufficient level. The fully‐formed holoenzyme can then degrade any remaining excess Rpt subunits, thereby regulating its own Rpt ring assembly. These two alternative mechanisms, degradation and sequestration of Rpt subunits, may help control the onset of chaperone‐mediated Rpt ring assembly, thereby promoting proper proteasome holoenzyme formation.

Support or Funding Information

R01GM127688
Cardiac Neuroanatomy and Chronotropic Modulation of the Adult Giant Danio Heart
Authors: Nelson Emma , Koch Alyssa , Lafontant Pascal J.
Published: April 2020

Studies in non‐mammalian models species have contributed significantly to our understanding of the biology and the nature of innervation in the heart. The giant danio (D. malabaricus) is a teleost fish species closely related to zebrafish, that is also capable of heart regeneration. We previously described the development and maturation of the giant danio (GD) heart. However, little is known about its innervation. We hypothesized that the pattern of innervation in the GD heart is anatomically and physiologically complex, and that the heart is responsive to physiological modulation similar to that seen in cyprinid fish and mammals. Using various neuronal markers and electron microcopy, we described the presence, distribution, and nature of nerves in the GD heart. Our study shows first that fine intrinsic cardiac nerve fibers are present throughout the heart chambers. Second, nerve soma and ganglia are highly concentrated at nerve plexuses located near the sinoatrial (SA) and atrioventricular (AV) junctions. However, the volume density of axonal processes_ projecting from ganglia located over the ventral aorta_ is highest over the corpus of the bulbus arteriosus. Third, using an ex vivo GD heart preparation, we found that the GD heart responded to both adrenergic and cholinergic agonists, in a manner that mirrors mammalian and teleost hearts. Taken together, our studies show that the GD heart displays complex patterns of innervation, and conserved cardiac physiological responses, and strongly suggest that the GD could be used as a viable model for investigating cardiac biology.

Support or Funding Information

• NIH R15 HD084262‐01, Martha C. Rieth Faculty Fellowship, Buehler Family Foundation
Employment of Different Waveform Shapes for Electrical Stimulation of the Vagus Nerve in Rats
Authors: Gomes Amanda Rodrigues Correa Frota , Subar Leonardo Guimarães , Ribeiro Raianne Stefane Dias , Brognara-Dias Fernanda , Dias Daniel Penteado Martins
Published: April 2020

The electrical stimulation of neural pathways has gained therapeutic relevance over the past decades. A wide range of clinical conditions benefits from vagus nerve stimulation (VNS), such as depression, epilepsy, heart failure, rheumatoid arthritis, cluster headache, traumatic brain injury, among others. Considering the relevance of experimental protocols aimed at a better understanding of mechanisms underlying the effects of VNS, proper adjustment of stimulation parameters such as frequency, pulse width, electric tension and waveform shape is crucial. The current study was intended to evaluate the effects of VNS on bradycardic response using three different waveform shapes. Wistar male rats were anesthetized with a ketamine/xylazine mixture and implanted with an electrode for electrocardiogram (ECG) recording and a bipolar stainless‐steel electrode into the cervical portion of the vagus nerve for VNS. Anesthetized animals had the ECG recorded and heart rate (HR) calculated on a beat‐to‐beat basis. Baseline HR was obtained during 5 minutes, followed by sessions of electrical stimulation of the vagus nerve. Taking into consideration the shape of the waveform used into VNS, rats were assigned into three groups: monopolar pulse (MP), depolarizing prepulse (DPP) and bipolar pulse (BP). Stimulation parameters were similar for all groups (frequency: 5 Hz; pulse width: 100 microseconds; electric tension: 1 V). Each VNS session lasted 30 seconds and sessions were 3 minutes apart. HR response during VNS was calculated to ensure the reliability of the preparation and was taken as the difference between average HR preceding VNS onset and maximum bradycardic response during the 30 seconds session. The magnitude of bradycardic response following the first VNS session was higher among DPP rats (Δ115 ± 23 bpm) when compared to MP (Δ37 ± 9 bpm) and BP (Δ83 ± 14 bpm) groups. Also, since our experimental setting contributes for better establishment of neural pathway longevity following exposure to electrical stimulation, the averaged bradycardic response among the three successive sessions of VNS was assessed. In a similar fashion to the findings regarding one single VNS session, it was observed a trend towards higher bradycardic response in DPP rats (Δ90 ± 32 bpm), when compared to MP (Δ56 ± 24 bpm) and BP (Δ70 ± 5 bpm) groups. From our findings, we can conclude that depolarizing prepulse waveform elicits a higher bradycardic response when VNS is conducted using parameters that match other waveform shapes. It is noteworthy to mention that the employment of VNS using techniques gentle (i.e. diminished damage) to neural pathways could contribute to nerve longevity.

Support or Funding Information

Barao de Maua University Center: 344/18; FAPESP: 2017/05163‐6
Effect of Intermittent Hypoxia on Ischemic‐Reperfusion Injury in Healthy Individuals
Authors: Jarrard Caitlin P. , Nagel Mercedes J. , Tanaka Hirofumi , Lalande Sophie
Published: April 2020

Brief periods of ischemia preceding an ischemic‐reperfusion injury attenuate the reduction in brachial artery endothelial function. It has remained unknown whether brief bouts of systemic hypoxemia would similarly mitigate the blunted vasodilatory response induced by an ischemic‐reperfusion injury. Therefore, the purpose of this study was to determine whether intermittent hypoxia protects against an ischemic‐reperfusion injury in young healthy adults. Ten healthy individuals, 7 men and 3 women (age: 24 ± 2 years, height: 176 ± 9 cm, weight: 77.1 ± 13.9 kg), participated in the study. Brachial artery endothelial function was assessed by flow‐mediated dilation before and after an ischemic‐reperfusion injury induced by a 20‐minute blood flow occlusion. The ischemic‐reperfusion injury was preceded by either intermittent hypoxia (Hyp) or intermittent normoxia (Norm). Both visits were separated by a period of seven days, whereas women who had regular menstrual cycles were scheduled in the early follicular phase. Intermittent hypoxia was created by titrating nitrogen into a breathing system to achieve an arterial oxygen saturation of 90%. Intermittent hypoxia consisted of three 4‐minute hypoxic cycles separated by 4‐minute normoxic cycles. As expected, intermittent hypoxia resulted in a lower arterial oxygen saturation (Hyp: 88 ± 4 vs. Norm: 98 ± 2%, p < 0.01), which was equivalent to a lower fraction of inspired oxygen (Hyp: 0.13 ± 0.04, Norm: 0.21 ± 0.03, p < 0.01). The reduction in flow‐mediated dilation resulting from the ischemic‐reperfusion injury was attenuated by intermittent hypoxia (Hyp: 5.9 ± 0.7 to 4.5 ± 0.7%, Norm: 6.0 ± 0.7 to 3.6 ± 0.7%, p = 0.07). Exposure to intermittent hypoxia did not affect mean arterial blood pressure (Hyp: 97 ± 7 to 99 ± 8 mmHg, Norm: 94 ± 8 to 95 ± 10 mmHg, p = 0.52) but significantly increased heart rate (Hyp: 63 ± 6 to 70 ± 7 bpm, Norm: 61 ± 7 to 61 ± 6 bpm, p < 0.01). Thus, intermittent hypoxia seems to prevent the reduction in flow‐mediated dilation induced by an ischemic‐reperfusion injury in young healthy individuals.
The Relationship of Anatomical Self‐Efficacy and Feelings of Burnout in First Year Medical and Dental Students
Authors: Ferriby Andrew , Schaefer Audra
Published: April 2020

Feelings of burnout in professional students have been associated with additional psychological morbidities and decreased academic achievement. Due to the large quantity of content that professional students need to learn during gross anatomy courses, in an increasingly condensed time frame, it is possible that participation in gross anatomy may be a contributing factor to feelings of burnout. There are currently no studies linking participation in gross anatomy courses and feelings of burnout in professional students. Therefore, this IRB approved study (protocol #2019‐0178) is seeking to determine if such an association exists. Both first year medical (n=172) and dental (n=40) students are invited to provide basic demographic information at the beginning of the semester and to complete the Oldenburg Burnout Inventory‐Student Version (OLBI‐S) and the Anatomical Self‐Efficacy Scale (ASES) at the beginning, middle and end of the semester they are enrolled in gross anatomy. Anatomical self‐efficacy is defined as a student’s judgement of their ability to execute anatomical tasks. Our hypothesis is that students with lower levels of anatomical self‐efficacy will experience higher levels of burnout. To anonymously track each students’ progression through the semester, students are provided a prompt at each measurement point, that generates a unique identification code. Data collection for the medical students is ongoing and statistical analyses will be conducted at the end of the fall 2019 semester. Data collection for the dental students will occur during the spring 2020 semester. Typical descriptive statistics of the medical and dental student cohorts will be produced. Within‐subjects ANOVA will allow for comparison of scores on both the self‐efficacy and burnout scales to identify whether there are significant changes within a group during a single semester. Between‐groups ANOVA will allow for comparison of self‐efficacy and burnout scores between medical and dental students. Hierarchical linear regression will be conducted to determine the predictive nature between independent variables (anatomical self‐efficacy, demographic variables), and the dependent variable (burnout).
Integrating Genetic Fine‐mapping in Outbred Rats with a Novel Human Tubule Fibrosis Model Identifies Sept8 as a Gene Involved in Cellular Structure and Kidney Fibrosis
Authors: Woods Leah Solberg , Prokop Jeremy , Keele Gregory , Holl Katie , He Hong , Littrell John , Deal Aaron , Valdar William , Garrett Michael
Published: April 2020

Chronic kidney disease (CKD), which can ultimately progress to kidney failure, is influenced both genetics and the environment. Genes identified in human genome wide association studies (GWAS) explain only a small proportion of the heritable variance, suggesting the need for additional model systems to identify CKD genes. We performed GWAS for urinary protein and serum biochemistries in male heterogeneous stock (HS) rats. Quantitative trait loci (QTL) were identified using a linear mixed effect model of imputed genotypes which accounts for unequal relatedness between rats. RNAseq and bioinformatics tools were used to identify candidate genes. We then developed a novel human tubule hypoxia induced fibrosis model to test these candidates. We identified two QTL for urinary protein and five for serum biochemistries. Protein modeling identified a missense variant within Sept8 as a proteinuria candidate. We demonstrate increased expression of Sept8 in the in vitro fibrosis model. We further demonstrate that SEPT8 localizes to the cilia in tubule cells under normal conditions and relocalizes outside the cilia after fibrosis, suggesting a role of SEPT8 in cellular organization and structure in response to environmental stress. In human biopsies, we show that SEPT8 expression levels increase with fibrosis levels, providing additional support for this gene in kidney fibrosis. This work suggests that integration of a rat genetic model with an environmentally induced cell culture system of tubule fibrosis has the power to elucidate aspects of the complex gene by environmental interactions that contribute to CKD risk.

Support or Funding Information

R01 DK088975 and R01 DK106386 (LSW) and NIH/NHLBI R01HL137673 (MRG)
Detection of Pesticide Residues in Fruits and Vegetables: Degradation and Removal by Ozonolysis
Authors: Relangi Praveen Kumar , Yakubu Momoh A.
Published: April 2020

We investigated effects of ozonization of the degradation and removal of pesticides residues in fruits and vegetables
New Metabolite Agonists and Allosteric Modulators for Understudied “Dark” G Protein‐Coupled Receptors
Authors: Isom Daniel , Kapolka Nicholas , Rowe Jacob , Taghon Geoff , Morgan Will
Published: April 2020

There are more than 100,000 known human metabolites, but less than 20 known metabolite‐sensing G protein‐coupled receptor (mGPCRs). This is surprising given that more than 800 GPCRs comprise the largest class of cell surface receptors in humans. Here, we confirm our hypothesis that there are more mGPCRs to be discovered by reporting new metabolite agonists and allosteric modulators for several understudied “dark” (GPR4, GPR65, GPR68, and HCAR3) and well‐known (ADRA2A, ADRA2B, MTNR1A, LPAR1, LPAR4, S1PR1, and S1PR2) receptors. We identified these interactions using our new ligand discovery technology, dynamic Cyan induction by Functional Integrated Receptors (dCyFIR), to screen a library of 320 metabolites against an exploratory panel of 300 GPCR‐Gα subunit coupling combinations covering 30 receptors. Our findings support the emerging view that numerous mGPCRs are distributed throughout the body to detect global (endocrine) and local (paracrine and autocrine) metabolite signals, and represent an untapped source of pharmacotherapeutic potential.

Support or Funding Information

This work was supported by R35GM119518, R01NS103906, and R03TR002908 to D.G.I.
Plant derived terpenes and their impact on the growth of the amphibian chytrid fungus Batrachochytrium dendrobatidis
Authors: Louros Andrew , Sun Patrick
Published: April 2020

Whole plant derivatives have been shown to have antifungal properties and inhibit the growth of Batrachochytrium dendrobatidis. Though the antifungal properties have been attributed to several elements within the whole plant extract, such as terpenes, few studies have examined their individual effects. This study characterizes the impact of terpenes on the settlement and propagation of B. dendrobatidis. Population density is characterized using well established absorbance procedure. Our results show that the terpenes, in low concentrations increase growth of B. dendrobatidis until a certain threshold.
WNK1 Regulates Cell Volume via Crowding‐Induced Phase Transitions
Authors: Boyd-Shiwarski Cary R. , Shiwarski Daniel J. , Weaver Claire J. , Beacham Rebecca T. , Griffiths Shawn E. , Subramanya Arohan R.
Published: April 2020

When challenged by hypertonicity, dehydrated cells recover their volume though regulatory volume increase (RVI). This process is thought to be initiated by an undefined intracellular sensing and transduction mechanism that responds to macromolecular crowding (Parker JC AJP 1993). In many cell types, RVI is mediated by the bumetanide‐sensitive cotransporter NKCC1, which is phosphorylated and activated by the WNK‐SPAK/OSR1 pathway. Reconciling the mechanism of hypertonic stress‐induced activation of NKCC1 has been challenging, as upstream WNK kinases such as WNK1, though required for bumetanide‐sensitive RVI (Roy A AJP 2015), should be inactivated by the strengthened intracellular chloride concentration caused by cell shrinkage (Piala AT Sci Signaling 2014). We describe our efforts to unravel this paradox using a combination of high‐speed live cell and fluorescence lifetime imaging, correlative light electron microscopy, gene editing, and biochemical approaches in cells. During hyperosmotic cell shrinkage and macromolecular crowding caused by reduced cell water, WNK1 dynamically concentrates into membraneless liquid droplets. These changes in WNK1 localization occur at native levels of expression and are visible within physiologic range (25 mOsm above isotonicity), triggering a cascade of condensed WNK1 autoactivation, droplet fusion, cytosolic exchange, and downstream SPAK/OSR1/NKCC1 phosphorylation within a 15 minute bumetanide‐sensitive RVI time course. When cells are rapidly switched from hypertonic to hypotonic media, the WNK1 condensates quickly dissipate, confirming their reversible nature and association with physiologic changes in cell size and intracellular crowding. An optogenetic screening assay that employs the PHR domain of Cry2 to trigger light‐induced crowding and phase separation revealed that the formation of WNK1 condensates requires its large intrinsically disordered C‐terminus, whose strong regional disorder tendency is highly conserved across evolution despite poor sequence homology. This disorder‐encoded phase behavior allows WNK kinases to react to a crowded cytosol, bypass the ionic strength dilemma, and restore cell volume via mass action. Thus, our findings indicate that in addition to their well‐appreciated role as chloride sensors, WNK kinases are molecular crowding sensors that phase separate to coordinate a conserved hyperosmotic stress response. While this evolutionarily conserved mechanism appears to be critical for WNK kinases to sense cell shrinkage and amplify the NKCC1 RVI signal, we propose that it also impacts diverse WNK‐dependent signaling processes important in mammalian epithelial and neuronal physiology.

Support or Funding Information

NIH R01DK098145, R01DK119252, K08DK118211, F32HL142229, P30DK079307
Role of Src homology/collagen adaptor protein p66Shc in Pyk2 translocation into mitochondria under Gq protein–coupled receptor stimulation
Authors: Dileepan Gayathri , Polina Iuliia , Cypress Michael , Suzuki Yuta , Adhikari Neeta , Jhun Bong Sook , O-Uchi Jin
Published: April 2020

Introduction

Mitochondrial Ca2+ (mtCa2+) uptake via a main mtCa2+ channel, mtCa2+ uniporter (MCU), mediates cell survival and death via its contribution of mitochondrial ATP synthesis and apoptotic signaling activation, respectively. Our group previously showed that Gq protein–coupled receptor (GqPCR) stimulation promotes activation of a tyrosine kinase named proline‐rich tyrosine kinase 2 (Pyk2), and its translocation from cytosol to the mitochondrial matrix, which leads to the tyrosine phosphorylation of MCU, and mtCa2+ overload, and activation of apoptotic signaling in the cardiomyocytes. However, it is still unclear how Pyk2 access from cytosol to mitochondrial matrix under GqPCR stimulation. The Src homology/collagen (Shc) adaptor protein, p66Shc, is also known to be capable to translocate to the mitochondria where it promotes increased ROS production and apoptosis. Importantly, p66Shc can bind to another tyrosine kinase Src, but its association to Pyk2 has not been well investigated.

Hypothesis

p66Shc serves as an important chaperon for Pyk2 translocation into the mitochondrial matrix.

Aim

to test whether p66Shc can associate with Pyk2.

Methods

Mammalian expression plasmids containing p66Shc or Pyk2 were transformed in to bacteria and purified. Overexpression of p66Shc and/or Pyk2 in HEK293T cells were performed by transient plasmid transfection. Whole cell lysates were collected and used for biochemical assay including Western blotting and co‐immunoprecipitation assay.

Results

We confirmed that transient transfections of the plasmids containing His‐tagged p66Shc and GFP‐tagged Pyk2 were successfully yield overexpression of these proteins in HEK293Tcells. Co‐immunoprecipitation assay using GFP antibody showed that p66Shc and Pyk2 forms complex in the whole cell lysates from cells co‐transfected with two plasmids.

Conclusion

p66Shc can bind Pyk2 and may contribute to the mechanism for Pyk2 translocation to mitochondria. Pur next plan is to test whether the removal (overexpression) of p66Shc blocks (Pyk2 transportation and prevent excessive calcium uptake by the MCU under GqPCR stimulation both in heterologous expression system and primary cardiomyocytes.

Support or Funding Information

Apart of this research was supported by the Lillehei Heart Institute Summer Research Fellowship (to G.D.), American Heart Association (AHA) 18CDA34110091(to B.S.J), NIH/NHLBI R01HL136757 (to J.O.‐U.), AHA 16SDG27260248 (to J.O.‐U.), and American Physiological Society (APS) 2017 Shih‐Chun Wang Young Investigator Award.
The Impact of Dietary Sodium on Sympathetic and Blood Pressure Reactivity Assessed using the Cold Pressor Test
Authors: McMillan Ronald K. , Babcock Matthew C. , Migdal Kamila U. , Robinson Austin T. , Watso Joseph C. , Stocker Sean D. , Wenner Megan M. , Farquhar William B.
Published: April 2020

High dietary sodium intake increases sympathetic and blood pressure (BP) reactivity in rodents, but it is not known if dietary sodium similarly affects reactivity in non‐hypertensive humans. Importantly, exaggerated sympathetic and BP responses are predictive of future hypertension risk.

PURPOSE

We hypothesized that high dietary sodium would increase sympathetic and BP reactivity in male and female adults. The cold pressor test (CPT) was used to assess reactivity.

METHODS

Twenty‐seven healthy, non‐hypertensive adults (16M/11F; age: 26±7yrs; BMI: 24.7±1 kg/m2; mean±SD) participated in a controlled feeding study that consisted of 10 days of low (LS: 1.0 g/day), recommended (MS: 2.3 g/day), or high (HS: 7.0 g/day) sodium diets, in random order with at least a one‐month washout between diets. Twenty‐four hour urine samples were collected and sodium excretion was calculated to ensure compliance on the respective diets. Muscle sympathetic nerve activity (MSNA) was directly assessed using peroneal microneurography and beat‐to‐beat BP was assessed using photoplethysmography at rest and throughout the CPT. The CPT was completed by having participants immerse their dominant hand in an ice slurry (3° to 5°C) for 2 minutes. Data were analyzed using two‐way repeated measures ANOVAs.

RESULTS

As expected, twenty‐four hour sodium excretion increased with increasing amounts of dietary salt (LS: 42±40, MS: 84±36, HS: 253±111 mmol/24 hours, main effect of diet, p<0.0001), with no apparent sex differences (p = 0.98). The CPT robustly increased MSNA, but there was no effect of diet (Δ MSNA burst frequency LS: 13±6, MS: 16±6, HS: 15±5 bursts/min, p = 0.23). Similarly, the CPT acutely increased systolic BP, but there was no effect of diet (Δ systolic BP LS: 18±11, MS: 20±12, HS: 18±11 mmHg, p = 0.44).

CONCLUSION

These data suggest that 10 days of high dietary salt intake does not affect MSNA or BP responses to the CPT in young, healthy, non‐hypertensive adults.

Support or Funding Information

Supported by NIH Grant 1R01HL128388
Postnatal loss of Cyp26a1 and Cyp26b1 in mice causes impaired retinoid homeostasis in multiple organs
Authors: Zhong Guo , Snyder Jessica , Hogarth Cathryn , LaFrance Jeffrey , Huang Weize , Topping Traci , Palau Laura , Ghiaur Gabriel , Duncan Kellyanne , Vaishnava Shipra , Czuba Lindsay , Isoherranen Nina
Published: April 2020

Retinoic acid (RA), the active metabolite of vitamin A (retinol), is essential to various biological processes such as maintenance of epithelia and regulation of cell division, proliferation and differentiation. Cyp26a1 and Cyp26b1 enzymes catalyze hydroxylation of RA and are believed to be responsible for RA clearance in all chordates. Global knockout of either Cyp26a1 or Cyp26b1 is embryonic lethal, demonstrating a critical role of Cyp26 enzymes in regulating RA signaling during fetal development. However, whether Cyp26 activity is required in postnatal life to maintain vitamin A homeostasis is still not clear. We hypothesized that postnatal loss of Cyp26a1 and Cyp26b1 would decrease RA clearance, and consequently alter tissue retinoid concentrations and cause retinoid toxicity. A tamoxifen‐inducible Cre‐loxP system was used to induce a conditional global knockout of Cyp26a1 and Cyp26b1 (double‐knockout; DKO). The knock‐out was induced in female and male mice with tamoxifen injections at 21 days postpartum and then these animals were monitored (body weight and condition) biweekly for 8 weeks. Animals were euthanized if they received a body condition score ≤ 2 or had lost > 15% of their pre‐tamoxifen injection body weight. Tissues were harvested for histological analysis and retinoid measurements. All WT animals were healthy and euthanized after the 8‐week follow‐up but 45% of male and 41% of female DKO animals were euthanized before the completion of the 8‐week follow‐up according to the criteria described above. Compared to WT mice, DKO animals showed significant decrease in weight gain. DKO mice had moderate to severe blepharitis and dermatitis with skin thickening and inflammation. Spleens of DKO mice were significantly enlarged and showed extramedullary hematopoiesis. Cell proliferation in the epidermis and in the epithelium of nonglandular stomach was increased in DKO animals. In general, retinoid homeostasis was altered in DKO animals. In comparison to WT animals, skin, spleen and liver RA concentrations were significantly increased in DKO mice while tissue retinol and retinyl ester concentrations were unchanged. In contrast, serum retinol and retinol binding protein 4 (RBP4) concentrations were significantly decreased in DKO mice with no change in RA concentrations. When DKO animals were dosed with exogenous RA (1mg/kg; i.p.), animals showed signs of toxicity and the exposure to RA was significantly greater in DKO animals than in WT animals. Collectively these data show that Cyp26a1 and Cyp26b1 are required for maintaining vitamin A homeostasis and retinoid signaling also in postnatal life.
Characterizing a Novel L‐Ascorbate Metabolic Pathway in Bacteria
Authors: Jermain Madison Danielle
Published: April 2020

L‐Ascorbate, commonly known as Vitamin C, is a naturally abundant carbon source produced in high quantities by plants. The involvement of L‐ascorbate as an antioxidant in processes within the body systems of mammals is well studied, yet L‐ascorbate can also function as a carbon source for some bacterial species. Ralstonia eutropha H16 (Cupriavidus necator H16) is a soil bacterium that stores excess carbon in bioplastic granules and catabolizes L‐ascorbate via a previously unknown metabolic pathway. Transcripts for eight genes were upregulated >50‐fold in R. eutropha cells grown with L‐ascorbate compared to fructose or succinate. Three encode homologs of enzymes in L‐lyxonate catabolism to α‐ketoglutarate. Based on annotated functions of the five remaining enzymes, a predicated pathway was hypothesized to generate a‐ketoglutarate from L‐ascorbate. Enzymes not homologous to the L‐lyxonate pathway enzymes were purified and characterized in vitro by collaborators. Mutant R. eutropha strains were generated, each lacking one of the eight genes, and displayed significant growth defects when grown with L‐ascorbate. Support for a novel pathway for L‐ascorbate catabolism increases our knowledge of related enzyme functions and the involvement of L‐ascorbate in the carbon cycle.
The Effects of Valve Leaflet Mechanics on Lymphatic Pumping Assessed Using Numerical Simulations
Authors: Munn Lance L. , Mei Yumeng , Baish James , Padera Timothy P. , Li Huabing
Published: April 2020

The lymphatic system contains intraluminal leaflet valves that bias lymph flow back towards the heart. These valves are present in the collecting lymphatic vessels, which generally have lymphatic muscle cells and can spontaneously pump fluid. Recent studies have shown that the valves are open at rest, can allow some backflow, and are a source of nitric oxide (NO). To investigate how these valves function as a mechanical valve and source of vasoactive species to optimize throughput, we developed a mathematical model that explicitly includes Ca2+ ‐ modulated contractions, NO production and valve structures. The 2D lattice Boltzmann model includes an initial lymphatic vessel and a collecting lymphangion embedded in a porous tissue. The lymphangion segment has mechanically‐active vessel walls and is flanked by deformable valves. Vessel wall motion is passively affected by fluid pressure, while active contractions are driven by intracellular Ca2+ fluxes. The model reproduces NO and Ca2+ dynamics, valve motion and fluid drainage from tissue. We find that valve structural properties have dramatic effects on performance, and that valves with a stiffer base and flexible tips produce more stable cycling. In agreement with experimental observations, the valves are a major source of NO. Once initiated, the contractions are spontaneous and self‐sustained, and the system exhibits interesting non‐linear dynamics. For example, increased fluid pressure in the tissue or decreased lymph pressure at the outlet of the system produces high shear stress and high levels of NO, which inhibits contractions. On the other hand, a high outlet pressure opposes the flow, increasing the luminal pressure and the radius of the vessel, which results in strong contractions in response to mechanical stretch of the wall. We also find that the location of contraction initiation is affected by the extent of backflow through the valves.
Survival of Mature Dendritic Cells Dependent on Clusterin Expression
Authors: Mendoza Roy Alexander , Malizia Alvaro Lopez , Garcia Guadalupe Ailin , Sabattê Juan Antilio
Published: April 2020

Clusterin is a multifunctional glycoprotein found in various tissues and body fluids. It is involved in a number of physiological and pathological processes such as apoptosis, protein homeostasis, cancer and Alzheimer’s disease. Although clusterin expression in myeloid cells has been reported, its influence in dendritic cell (DC) function has yet to be analyzed. Therefore, the purpose of our experiment was to determine the role of clusterin in DC survival. First, we analyzed whether activation of human monocyte‐derived DCs resulted in clusterin production. This was done by culturing cells for 48 hours with or without LPS (10 ng/ml). Clusterin was analyzed by western blot and quantified by ELISA. A basal expression level of clusterin was observed in unstimulated DCs, while an increase in production was noted upon activation of these cells by LPS: clusterin concentration in cell supernatant was 0.04 ± 0.1 vs 7.47 ± 2.14 ng/ml (control vs LPS, mean ± ES, n=5, p<0.01 vs unstimulated cells). In order to understand the dynamics of production after activation, we constructed a production curve measuring the level of clusterin post stimulation over a period of four days. A peak in the production of intracellular and secreted clusterin was noted 48 – 72 hours post stimulus. Finally, a knockdown (KD) strategy using a lentiviral gene silencing protocol that delivered a clusterin and scramble (SCR) shRNA was used to determine the role of clusterin in DC function. Two lentiviral combinations were used due to their ability to knockdown >85% of clusterin expression, as evaluated by western blot (in HeLa cells) and rtPCR (DCs). Puromycin was used as a selection drug and a Ficoll gradient was used to separate live and dead cells. We found that silencing clusterin expression (CLU‐KD) resulted in a massive death of DCs upon stimulation with LPS, as evaluated by anexin‐V/propidium straining: 63.7 ± 11.8, 19.7 ± 4.9, and 25.2 ± 6.5% of dead cells, for LPS‐stimulated CLU‐KD DCs, LPS‐stimulated scramble DCs, and unstimulated DCs, respectively (mean ± ES, n=4, p<0.01 CLU‐KD vs scramble or unstimulated DCs). These observations uncover a novel function of clusterin as a key player in the control of the adaptive immune response.

Support or Funding Information

Roy Mendoza was supported by the grant LA Basin Minority Health and Health Disparities Research Training Program (MHRT) T37MD001368 from the National Institute on Minority Health and Health Disparities, National Institute of Health.
Liver fibrosis and biliary proliferation are enhanced in the rat model of inflammatory bowel disease
Authors: O’Brien April , Chen Lixian , Cromer Walter , Alpini Gianfranco , Glaser Shannon , Zawieja David
Published: April 2020

Background

Patients who suffer from Inflammatory Bowel Disease (IBD) often experience concurrent conditions such as lymphatic dysfunction and bone loss. Additionally, liver and biliary abnormalities are frequently observed, including primary sclerosing cholangitis (PSC), autoimmune hepatitis, and cholelithiasis. The pathogenesis is unknown but is presumed to be secondary to chronic inflammation, which drives intrahepatic inflammation and fibrogenesis. The lymphatic system, via transport of inflammatory stimuli, is a likely route by which the regional inflammatory changes in the bowel effect distal organs such as the liver. Therefore, the aim of this study was to evaluate changes in biliary damage and senescence, ductular reaction (DR), and liver inflammation and fibrosis in an experimental rat model of inflammatory bowel disease.

Methods

Studies were performed in male rats receiving a 2,4,6‐trinitrobenzene sulfonic acid (TNBS) enemas at 30mg/kg of body weight, once a week, for 4 weeks. Lymph, livers, isolated cholangiocytes, and hepatic stellate cells were collected. Ductular reaction was evaluated by immunohistochemistry (IHC) for CK19 in paraffin embedded liver sections and cellular senescence was determined by SA‐β‐gal staining. Gene expression was measured using qPCR for p16, CCL2 and p21 in total liver and laser‐captured microscopy (LCM) isolated cholangiocytes. Liver fibrosis was assessed via Sirius Red staining and qPCR for the following genes; α‐SMA, TGFβ‐1, FN‐1, and collagen1a1 in total liver, HSCs, and pure cholangiocytes. To determine the level of inflammation, IHC for CD68 and MHCII was performed, while gene expression was measured using qPCR for inflammatory/senescence markers (IL‐1β, IL‐6, IL‐33 and TNF‐α) in total liver and LCM isolated cholangiocytes. Immunofluorescence was used to visualize the expression of TLR4 in the intact bile ducts of total liver sections. Lymph was used for proteomic analysis post‐instillation at peak inflammation, to identify potential inflammatory signals that may precipitate the changes that were noted.

Results

Rats treated with TNBS had increased DR, collagen deposition, and fibrotic gene expression in both cholangiocytes and HSCs; enhanced liver inflammation with increased CD68 positive macrophages in liver sections; increased biliary senescence and biliary expression of senescence markers; and increased biliary expression of TLR4. There was a strong correlation between the increase in inflammatory pathways in the lymph from IBD animals, with marked increases in pathways associated with bacterial translocation and lipid metabolism.

Conclusion

We observed increased biliary ductular reaction that was associated with increased liver inflammation/fibrosis similar to the co‐morbidity of PSC reported in IBD patients.

Support or Funding Information

NIH RO1 DK110035
6,7‐dihydroxy‐2,4‐dimethoxy phenanthrene isolated from Dioscorea batatas peel suppresses microglia‐associated neuroinflammation in vitro
Authors: Yun Hyun Seok , Lim Ji sun , Oh Jisun , Kim Jong-Sang
Published: April 2020

Inflammation occured in the neural tissue is a cause of neurodegenerative diseases. Our previous study showed that the ethanolic extract of Dioscorea batatas Decne peel promoted antioxidant mechanism in colonic epithelial cells and attenuated inflammatory response in activated macrophages. Recently, 6, 7‐dihydroxy 2, 4‐dimethoxy phenanthrene (DDP) has been identified as one of the bioactive compounds having antioxidative and anti‐inflammatory activities. The current study aimed at examining the neuroprotective potential of DDP by staving off neuroinflammation. Our data demonstrated that (1) DDP decreased glutamate‐induced murine hippocampal neuronal cell death; hlowever, there was no significant increase in ARE‐luciferase activity, (2) DDP decreased the expressions of nuclear NF‐κB and its downstream proteins in BV2 murine microglial cells and thereby lowered NO production and cytokine secretion, and (3) DDP increased the production of antioxidant enzyme, heme oxygenase 1(HO‐1) in BV2 murine microglial cell. These results indicate that DDP suppressed microglial activation which generally causes neuroinflammation. In consideration of our observations implying that DDP may not directly stimulate the antioxidant mechanism in neuronal cells for its in vitro neuroprotective effect, DDP would possibly enhance neuronal cell survival due to its anti‐neuroinflammatory capability in microglia. The interaction between neuronal cells and microglia cells in culture as well as under physiological conditions awaits further study.
Bioactive properties of protein hydrolysate of cottonseed byproduct: antioxidant, antimicrobial, and angiotensin‐converting enzyme (ACE) inhibitory activity
Authors: Egea Mariana Buranelo , de Oliveira Filho Josemar Gonçalves , Lemes Ailton Cesar , Valencia-Mejia Erika , Fernandes Katia Flavia , de Figueiredo Sousa Heloisa Alves , da Silva Edilsa Rosa , Dyszy Fábio H.
Published: April 2020

Cottonseed meal is a byproduct generated in large amounts resulting from the process of extracting cottonseed oil. The aim of this study was to investigate the cottonseed byproduct hydrolysate produced with Alcalase® (A), Neutrase® (N), or Flavourzyme® (F), and thus, evaluate its the antioxidant activity and angiotensin‐converting enzyme (ACE) inhibitory activity, as well as ability to inhibit the strains of Staphylococcus aureus, and Escherichia coli. The protein hydrolysates (PH) were obtained using thermal pretreatment (CT – control and HT – thermal pretreatment) and enzymatic hydrolysis of protein extract obtained of cottonseed byproduct. The decreasing order of the degree of hydrolysis of protein hydrolysate: HT‐A>CT‐A>HT‐N>CT‐N>HT‐F>CT‐F. The DPPH scavenging activity of the hydrolysates obtained from the Alcalase®, Neutrase®, and Flavourzyme® treatments at the end of the reaction (100 min) were 95.44%, 78.10%, and 62.94% for the HT hydrolysates, respectively, and 88.60%, 70.69%, and 62.06% for the CT hydrolysates, respectively. The ferric reducing antioxidant power of the hydrolysates obtained from the Alcalase®, Neutrase®, and Flavourzyme® treatments at the end of the reaction were 0.47, 0.44, and 0.34 mg TE/mg sample for the HT hydrolysates, respectively, and 0.38, 0.43, and 0.28 mg TE/mg for the CT hydrolysates, respectively. The results of the growth inhibition of S. aureus obtained from the Alcalase®, Neutrase®, and Flavourzyme® treatments at the end of the reaction were 32.38%, 16.66%, and 22.35 for the HT hydrolysates, respectively, and 28.35%, 16.68%, and 17.85% for the CT hydrolysates, respectively. The heat‐pretreated hydrolysates were able to inhibit the growth of S. aureus. The growth inhibition of E. coli was lower when compared to that of S. aureus. The growth was inhibited from 13 to 18 % and was observed for the HT hydrolysates produced using Alcalase® after 20 min of reaction. The hydrolysates showed ACE inhibition of 74 to 99 %. The hydrolysate produced using Alcalase® with thermal treatment showed higher antioxidant activity and ACE inhibition (99.5%) than other enzymes. In this study, it was possible to obtain PH with different bioactivity properties using adequate processing by successfully choosing proteolytic enzymes and thermally pretreating proteins from the cottonseed byproduct.

Support or Funding Information

This work was supported of the FAPEG (Process 201610267000530), CAPES (001), CNPq (06/2016), IFB (030/2016) and IF Goiano (Process 23218.000788/2018‐41).