bluuebunny/crossref_metadata_2025_split huggingface dataset:
CDK1‐dependent Phosphorylation of the Tumor Suppressor Phosphatase, PHLPP1, Regulates the Mitotic PHLPP1 Interactome
Authors: Kawashima Agnieszka Grzechnik , Wong Cassandra , King Charles C. , Gingras Anne-Claude , Newton Alexandra 
Published: April 2020

PH domain Leucine Rich Repeat Protein Phosphatase 1 (PHLPP1) is a tumor suppressor originally discovered for its ability to directly dephosphorylate and inactivate the pro‐survival kinase Akt, a key transducer of growth factor signaling. A number of other PHLPP1 targets have been identified, but still little is known about the molecular mechanisms governing the function and regulation of PHLPP1 itself. Here we report that PHLPP1 is hyperphosphorylated during mitosis in a CDK1‐dependent manner, and that this hyperphosphorylation regulates its interaction with mitotic proteins. Specifically, we show that PHLPP1 undergoes an electrophoretic mobility shift in mitotic cells that is lost with lambda phosphatase treatment, and is prevented by CDK1 inhibition. This mobility shift can be recreated in vitro using recombinant CDK1‐Cyclin B. Mass spectrometry and biochemical analysis reveals that these phosphorylations modify the N‐terminus of PHLPP1, a functionally uncharacterized region. A proximity dependent biotin identification (BioID) interaction screen revealed that mitotic PHLPP1 interacts with components of the mitotic spindle apparatus and the kinetochore. Additionally, the data suggest that the N‐terminus is required for the dissociation of PHLPP1 from interphase scaffolds, such as Scribble, during mitosis. During mitotic exit, PHLPP1 protein levels decrease, suggesting that PHLPP1 is degraded during mitotic exit. This correlates with an increase in Akt Ser473 phosphorylation, a validated cellular target of PHLPP1. Our data are consistent with a model in which phosphorylation of PHLPP1 during mitosis regulates binding to its mitotic partners and allows proper passage through mitosis. Reversible protein phosphorylation, orchestrated by kinases and phosphatases, plays a role in controlling proper progression through mitosis, which is essential as errors in mitosis can result in aneuploidy, a hallmark of cancer. The finding that PHLPP1 binds mitotic proteins in a cell cycle and phosphorylation‐dependent manner may have relevance to its tumor suppressive function.Support or Funding InformationThis work was supported by NIH R35 GM122523, T32 GM007752, & The Frontiers of Innovation Scholars Program.
Do Second Messengers of Neuroendocrine Factors Alter Aquaporin Expression in Malpighian tubules of the Mosquito, Aedes aegypti?
Authors: Picinic Britney N. , Paluzzi Jean-Paul , Donini Andrew 
Published: April 2020

The mosquito Aedes aegypti is considered one of the most dangerous disease vectors because it can transmit several arboviral diseases including Dengue, Chikungunya, Zika virus, and yellow fever. The larvae are aquatic typically inhabiting hypotonic freshwater whereas the adults are terrestrial. Hence, the larvae are usually faced with the osmoregulatory challenge of eliminating excess water from body fluids while adults usually conserve water, that is, with the exception of females when they take a blood meal, at which time they too need to eliminate excess water. Osmoregulation is accomplished, in part by regulating water flux across epithelia of organs such as the midgut, Malpighian tubules (MTs), hindgut and, in the case of larvae, the anal papillae. The MTs with the hindgut are functionally analogous to the renal tubes of mammalian kidneys where urine is produced; however, the mechanisms of urine production are different. In MTs, urine production is accomplished by ion secretion driven by an apical V‐type H+‐ATPase and water is obliged to follow by osmosis. There is a plethora of literature which describes the regulation of MTs fluid secretion in insects but these studies mostly describe effects on ion transport. Little is known about the regulation of water transport across the epithelium of the MTs. The mosquito, A. aegypti, possesses six aquaporin (water transporter) genes designated AaAQP1 through 6, which are expressed in the MTs and transcript abundance is affected by blood feeding in adult females. Furthermore, AaAQP5 mediates water flux across larval MTs whereas AaAQP1 is expressed in tracheolar cells of adult MTs. In this study we have begun to examine if and how known second messengers of neuroendocrine factors influence AaAQP protein expression in the MTs of A. aegypti with western blots. Thus far we have found that in MTs of larvae incubated with cAMP, AaAQP1 protein expression is higher than unstimulated controls but AaAQP4 levels are unaffected while both AaAQP1 and AaAQP5 expression is not affected by cGMP also in comparison to unstimulated controls. In larvae and adult mosquitoes, cAMP is a second messenger of diuretic hormones that stimulate urine production whereas cGMP is the second messenger of anti‐diuretic hormones, including CAPA peptides, which inhibit fluid secretion at physiological doses. In heterologous expression systems, AaAQP1 and AaAQP5 transport water whereas AaAQP4 is an entomoglyceroporin which transports solutes like trehalose preferentially over water. We conclude that stimulatory hormones of MTs facilitate fluid secretion at least in part by increasing the basal expression of AaAQP1 and that inhibitory hormones may not affect basal AaAQP expression; however, effects of these factors on stimulated MTs and on the expression of the other AaAQP genes is the subject of continuing work.Support or Funding InformationNSERC‐Discovery RGPIN‐2018‐05841
Chronic Intermittent Hypoxia Enhances GABAergic and Glycinergic Signaling in NTS Neurons
Authors: Jia Shuping , Farmer George E. , Cunningham Joseph Thomas 
Published: April 2020

In our past studies, spontaneous miniature IPSPs (mIPSCs) recorded from caudal NTS neurons surrounding the tractus solitarius appeared to be mainly mediated by GABA in normal rats. However, the averaged rise time and decay time are significant shorter in NTS neurons from rats after 7 days Chronic Intermittent Hypoxia (CIH) exposure as compared to normoxic, control rats (p<0.001). This study test whether CIH alters evoked inhibition in the NTS by recruiting a glycine mediated mechanism. The inhibitory postsynaptic currents(eIPSCs) evoked by electrical stimulation of the tractus solitarius were examined with whole cell patch‐clamp recordings on NTS second‐order neurons identified by DiA labeling of carotid bodies in a horizontal brainstem slice preparation from CIH exposed and control rats. All experiments were performed in the lateral NTS at the level of the area postrema. The results indicate that NTS neurons from the CIH rats had variable peak amplitudes from 67.6 to 617.28 pA compared with neurons from controls (47.15 to 284.07 pA). CIH significantly increased eIPSC amplitudes (33 neurons in CIH: −189.84 ± 22.76 pA vs 34 neurons in control: −124.3 ± 11.63 pA, p<0.05). This was accompanied by faster eIPSC kinetics (max rise slope of CIH: −150.54 ± 13.47 pA/ms vs control: −106.04 ± 8.82 pA/ms, p<0.01: and max decay slope of CIH: 53.24 ± 1.67 pA/ms vs control: 47.58 ± 1.41 pA/ms, p=0.01) that were significantly increased in neurons from CIH rats as compared to controls. To discriminate among glycine, GABA, or dual eIPSCs, we applied 3 μm strychnine (glyR antagonist) or 25 μm gabazine (GABAAR antagonist) in the presence of 10 μm CNQX (a competitive AMPA/kainate receptor antagonist). Baseline eIPSCs were recorded and either gabazine or strychnine was added to the perfusion solution to differentiate GABA‐mediated current and glycine‐mediated current. Of the 12 neurons from control rats, 7 neurons were gabazine‐sensitive, 1 neuron was strychnine‐sensitive, and 4 neurons had both GABA‐mediated and glycine‐mediated currents. In the 13 neurons from CIH rats, there were 4 gabazine‐sensitive neurons, 3 strychnine sensitive neurons and 6 neurons sensitive to both gabazine and strychnine. The glyR‐mediated eIPSCs expressed faster rise and decay kinetics. In NTS neurons from normoxic control rats, most eIPSCs appear to be GABA mediated while neurons from CIH rats demonstrated glycine and mixed eIPSCs. Our findings suggest that evoked glycinergic signaling in the NTS may be enhanced by seven days of CIH exposure.Support or Funding InformationThis work was supported by P01 HL088052
Optogenetic activation of preBötzinger Complex cells alleviates respiratory depression by opioids
Authors: Rousseau Jean-Philippe , Montandon Gaspard 
Published: April 2020

RationaleOpioids are extensively used for their analgesic properties but present a variety of unwanted side effects, including tolerance, dependence and respiratory depression. The analgesic effect of opioids is due to activation of μ‐opioid receptors (MOR) in the central nervous system and no treatments are currently available to prevent respiratory depression without reducing their analgesic properties. The neural circuits and mechanisms regulating respiratory depression, sedation, and analgesia by opioids often overlap, therefore making challenging the identification of the mechanisms regulating respiratory depression. We previously showed that neurons expressing neurokinin‐1 receptors located in the preBötzinger Complex (preBötC) also co‐express the peptide somatostatin (SST) and MORs. Neurokinin‐1 receptors are preferentially inhibited by opioids and play an essential role in mediating opioid‐induced respiratory depression. The role of SST preBötC cells in regulating respiratory depression by opioids is unknown.ObjectiveHere, we tested the hypothesis that optical stimulation of SST‐expressing preBötC cells will prevent or reverse respiratory rate depression by opioids.MethodsUsing a Cre‐loxP recombination approach, we injected stereotaxically, in SST Cre recombinase mice the adeno‐associated virus containing the gene cassette of the excitatory channelrhodopsin‐2 ChETA flanked between loxP sites. A three week recovery period is usually enough to allow ChETA expression in SST cells. A 200 μm optical fiber was then positioned above the preBötC for laser stimulation with blue light (wavelength: 480 nm). Respiratory rate was measured in anesthetized mice with electromyographic recordings of the diaphragm and genioglossus muscles activity. Once breathing was stable (>30 min), the clinically‐relevant μ‐opioid drug fentanyl (1mg/kg) was administered by intramuscular injection. About 15 min after fentanyl injection, SST preBötC cells were stimulated using laser stimulations (frequency: 20Hz, duration of stimulation: 300ms).ResultsOur preliminary data showed that fentanyl depressed respiratory rate by about 40% in 15 min consistent with our previous studies using mice. Stimulation of SST‐expressing preBötC cells increased breathing rate back to baseline level when the laser was on. Once the laser stimulation was stopped, respiratory depression continued to progressively decrease.ConclusionSST preBötC may constitute cell targets to prevent respiratory rate depression by opioids. These results may help identify the cells mediating respiratory depression by opioids, an essential step toward the development of therapeutic targets to reduce the risk of opioids overdose and associated mortality.Support or Funding InformationSupported by CIHR and FRQS
The Function and Regulation of Chloroplast Targeting of AZI1, a Key Factor in Plant Systemic Immune Signaling
Authors: Speed DeQuantarius Javon , Greenberg Jean T. 
Published: April 2020

Recognition of microbes and microbe‐associated molecular patterns by plant cell receptors induces signaling cascades that prime broad‐spectrum defense responses to suppress subsequent infections. Primed plants can activate stronger defense responses more quickly than unprimed plants when challenged by pathogens and other stresses. Systemic priming requires the long‐distance movement of signals produced during initial exposure events. Distinct responses are induced depending on whether roots are colonized by specific commensal microbes or if leaves are infected by certain pathogens. The protein AZI1, a member of the lipid transfer protein superfamily, is essential for both root‐stimulated and leaf‐stimulated defense priming. AZI1 is also needed for uptake and mobilization of azelaic acid (AZA). A defense signal derived from chloroplast lipid oxidation, the oxylipin AZA stimulates priming when applied to leaves or roots.Functional AZI1‐GFP traffics among chloroplast outer envelope membranes (OEMs), ER, PM, and intercellular channels called plasmodesmata. The targeting of AZI1 to chloroplast OEMs requires a newly described bipartite protein targeting signal featuring an N‐terminal hydrophobic domain and an internal region called the proline‐rich region. AZI1’s association with chloroplasts and plasmodesmata is suggestive of a pathway by which AZI1 may act in the intercellular mobilization of a chloroplast‐derived lipid signal(s) such as AZA. Consistent with this hypothesis, AZI1 increasingly targets to chloroplasts under infection conditions. This infection‐induced chloroplast accumulation requires the kinase MPK3, which, like AZI1, is essential for priming defenses and is reported to phosphorylate AZI1 in vitro.To better understand the molecular basis of plant immunity, it is essential to assess the location and movement of chloroplast‐derived priming signals; here, I report on the function of AZI1’s localization to chloroplasts and the regulation of this chloroplast targeting using the model plants Arabidopsis thaliana and Nicotiana benthamiana. A more thorough understanding of priming will have profound agricultural benefits via genetic engineering or breeding of crops. Enhanced priming will boost immunity only during infection and would reduce fitness costs associated with constitutively active defenses.Support or Funding InformationThis work is supported by the NSF, the MCB training grant (T32 GM007183), the Ford Predoctoral Fellowship, and the HHMI Gilliam Fellowship for Advanced Study.
High salt activates NLRP3 inflammasome in antigen presenting cells via ENaC to promote salt‐sensitive hypertension
Authors: Pitzer Ashley L. , Barbaro Natalia R. , Aden Luul , Ray Evan , Kleyman Thomas , Kirabo Annet 
Published: April 2020

Considerable evidence demonstrates that excess dietary sodium (Na+) is a major independent risk factor for hypertension and CVD in both hypertensive and normotensive subjects. Approximately 50% of hypertensive patients are salt‐sensitive, and reducing dietary sodium (Na+) decreases both blood pressure and CVD risk. The precise mechanisms of how salt leads to hypertension are still not well defined. The amiloride‐sensitive epithelial Na+ channel (ENaC) in non‐epithelial cells have been found to play a role in blood pressure regulation. Recently, our lab has shown that dendritic cells (DCs) in response to increases in extracellular [Na+] exhibit an ENaC‐dependent activation of NADPH‐oxidase, superoxide production, reactive isolevuglandin (IsoLG)‐protein adduct formation, and cytokine secretion which promote hypertension. In this study, we hypothesized that the NLRP3 inflammasome in antigen presenting cells (APCs) mediates salt‐sensitive hypertension through an ENaC‐dependent mechanism. To test this hypothesis, we cultured mouse splenocytes in normal‐salt or high‐salt media with or without co‐treatment with the ENaC inhibitor, amiloride (20 μM). Using flow cytometry, we found that high salt increased both monocyte and DC production of IL‐1β, which was confirmed through an ELISA assay detecting release of IL‐1β (2.131 ± 0.733 vs 12.75 ± 1.108 pg/mL, p<0.0001) into the culture media. Treatment with amiloride prevented production of IL‐1β (12.75 ± 1.108 vs 1.905 ± 0.3495 pg/mL, p<0.0001) in both monocytes and DCs. To confirm our in vitro data, we treated salt‐sensitive mice on a 129‐SvJ background with a high‐salt diet (4% NaCl) for 28 days with or without ENaC inhibitor amiloride (1mg/kg/day in drinking water) or NLRP3 inflammasome inhibitor MCC950 (10mg/kg i.p.). Mice treated with amiloride or MCC950 developed blunted hypertension (120.4 ± 2.99; 101.0 ± 3.74) compared to the high‐salt treated controls (140.5±3.98). Mice treated with amiloride also exhibited less expression of NLRP3, pro‐IL1b, and IsoLGs in both DCs and monocytes. Interestingly, the MCC950 treated mice only exhibited decreased pro‐IL1b but not NLRP3 expression or IsoLG production. Using the deoxycorticosterone acetate and 1% salt diet (DOCA‐salt) model, we found similar increases in expression of NLRP3, pro‐IL1b, and IsoLGs in DCs and monocytes as the 4% NaCl high salt diet fed mice. Treatment of the DOCA‐salt mice with the IsoLG scavenger 2‐HOBA (1g/L) attenuated expression of NLRP3, pro‐IL1b, and IsoLGs in both DCs and monocytes. Our findings suggest a role for ENaC‐dependent NLRP3 inflammasome activation in APCs in response to a high‐salt diet, which may represent a promising approach to treatment of salt‐induced hypertension.Support or Funding InformationK01HL130497, R01HL147818, T32HL144446
Characterization of 1700029J07RIK, a Novel Nuclear Membrane Associated Protein that is Induced During Skeletal Muscle Atrophy
Authors: Waddell David , Lynch Sarah 
Published: April 2020

Skeletal muscle atrophy is the loss of muscle mass that results from myriad physiological conditions including denervation, corticosteroid exposure, and aging. 1700029J07RIK is a putative novel gene that is induced in skeletal muscle in response to denervation and is differentially regulated in Muscle RING Finger 1 (MuRF1) knockout mice compared to wild‐type mice. A previous microarray analysis of muscle tissue isolated from wild‐type control mice and MuRF1 knockout mice revealed that the expression of the 1700029J07RIK gene is unchanged at 3 days post‐denervation, while expression is significantly induced in wild‐type mice at 14 days post denervation but remains unchanged in the MuRF1‐null animals. It has been well established that muscle cells go through a distinct developmental timeline in which they proliferate as single cell myoblasts and then differentiate and fuse into multinucleated myotubes. Quantitative PCR was used to determine that the 1700029J07RIK gene is expressed in proliferating cells and increases in expression as muscle cells differentiate. In addition, the 1700029J07RIK cDNA was cloned from muscle cells and fused with green fluorescent protein and visualized by confocal fluorescent microscopy revealing an interesting cytoplasmic and nuclear membrane localization of the 1700029J07RIK protein in proliferating muscle cells that is not evident in differentiated muscle cells. Utilizing cellular fractionation, it was determined that 1700029J07RIK is localized to both the cytoplasmic and nuclear fractions in proliferating cells, but then appears to translocate into the nucleus as muscle cells differentiate. Interestingly, the 1700029J07RIK protein appears to undergo dramatic post‐translational modification as relocation to the nuclear compartment progresses. Finally, it was observed that overexpression of 1700029J07RIK resulted in inhibition of muscle cell differentiation and attenuation of the ERK1/2 branch of the MAP Kinase signaling pathway. While there is virtually nothing known about this novel gene in the context of skeletal muscle, the results presented here suggest 1700029J07RIK may play a role in muscle cell differentiation and in the skeletal muscle atrophy pathway.Support or Funding InformationThe work described in this study was supported by UNF Transformational Learning Opportunity grants, Academic Affairs Faculty Enhancement grants, a Dean’s Leadership Council Fellowship Award, a UNF Foundation Board Grant, and research start‐up funds from the University of North Florida.
The Effect of Acute Passive Heating on Microvascular Oxygen Delivery, Exercise Tolerance and Recovery
Authors: Didier Kaylin D. , Alexander Andrew M. , Hammer Shane M. , Barstow Thomas J. 
Published: April 2020

The purpose of this study was to determine the effect of 1 bout of passive heating on oxygen delivery during knee extension exercise and neuromuscular recovery. We tested the hypotheses that: 1) 1 bout of passive heating would result in a decrease in the diffusive oxygen delivery (total‐[heme]) and an increase in the perfusive oxygen delivery (deoxy‐[heme]) in the exercising muscle, 2) peripheral fatigue would recover at a faster rate post heating and 3) passive heating would increase time to exhaustion.MethodsTwo men (183.9 ± 5.6 cm; 74.2 ± 10.8 kg) and two women (160.5 ± 0.7 cm; 53.8 ± 0.3 kg) performed intermittent isometric knee extension tests to exhaustion at 40% MVC pre and post 1 bout of passive heating. Absolute concentrations of deoxy‐[heme] (perfusive oxygen delivery) and total‐[heme] (diffusive oxygen delivery) of the vastus lateralis muscle were measured continuously via frequency‐domain multi‐distance near‐infrared spectroscopy (OxiplexTS, ISS) during exercise. Neuromuscular measurements of central (as measured by maximal voluntary contraction force, MVC; and voluntary activation, VA) and peripheral fatigue (as measured by potentiated twitch force, Qtw) were made every 30 s pre exercise and immediately after task failure for a total of 6 sets each. The passive heating protocol consisted of immersion up to the shoulder in a 40°C hot tu b until rectal temperature reached 38.5°C or increased by 1°C for 60 minutes.ResultsTime to exhaustion for pre and post passive heating was 553 ± 296 sec and 808 ± 491 sec, respectively (p=0.227), but 3 of the 4 subjects showed an increase in time to exhaustion post heating. From baseline to the last 60 seconds of exercise there was no significant difference in the increase in deoxy‐[heme] for pre (47.5 ± 30.8 μM) and post passive heating (38.5 ± 21.5 μM; p=0.526). Likewise, the increase in total‐[heme] during exercise was not significantly different following passive heating (p=0.117) (pre and post passive heating Δ total‐[heme] was 20.8 ± 19.8 μM and 8.3 ± 13.4 μM, respectively). There was no difference in baseline MVC pre to post heating (p=0.841), but MVC was significantly decreased following exercise for both pre and post passive heating (p<0.001). % change in VA from pre to post exercise was not different between pre and post heating (p=0.892). Qtw was not significantly different post exercise between pre to post heating. However, Qtw began to recover faster post compared to pre heating following exercise to exhaustion (30 sec vs. 90 sec, respectively).ConclusionThe trend of increased time to exhaustion after 1 bout of passive heating cannot be explained by increased perfusive or diffusive oxygen delivery. Passive heating led to a faster onset of recovery of peripheral fatigue (Qtw) following exercise to exhaustion, but the indices of central fatigue were not different pre vs. post heating.