bluuebunny/crossref_metadata_2025 huggingface dataset:
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 Information

NSERC‐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 Information

This 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

Rationale

Opioids 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.

Objective

Here, we tested the hypothesis that optical stimulation of SST‐expressing preBötC cells will prevent or reverse respiratory rate depression by opioids.

Methods

Using 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).

Results

Our 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.

Conclusion

SST 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 Information

Supported by CIHR and FRQS
Stability of Venom Collagenase Enzyme Activity and Effect of Order of Reactants with Inhibition by NNGH
Authors: Price Joseph A.
Published: April 2020

Earlier work had revealed complexities in enzyme inhibitor activity with snake venoms in reactions with a gelatin substrate (collagenase). To begin to clarify technical issues which could be in part related to enzyme stability or mechanism, baseline gelatinase activities of venom from three snake species: Agkistrodon contortrix contortrix, Crotalus atrox and Cerastes cerastes, were measured at room temperature using EnzChek® Gelatinase/Collagenase Kit E12055. A. c. contortrix and C. atrox were similar while C. cerastes venom showed the lowest baseline activity. Initial progress of reaction data with additional Selwyn plot analysis demonstrated enzymatic stability over a wide range of substrate concentrations and reaction times under the conditions tested. Next, because the order of reactants in enzyme inhibition reactions will affect results depending on the mechanism of inhibition, an hydroxamate inhibitor of interest was tested. The inhibitory effect of NNGH (N‐Isobutyl‐N‐(4‐methoxyphenylsulfonyl)glycyl hydroxamic acid) on venom gelatinase activity was not affected by enzyme pretreatment with inhibitor prior to the gelatinase reaction. These results will be useful to base later mechanistic studies to understand reaction kinetics of snake venom enzyme inhibition, which could lead to alternative treatment modalities of envenomation.
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 Information

This 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 Information

K01HL130497, 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 Information

The 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.

Methods

Two 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.

Results

Time 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).

Conclusion

The 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.
The inhibition of AGTR1 and Aromatase as a new potential therapeutic strategy for Glioblastoma treatment.
Authors: Panza Salvatore , Gelsomino Luca , Russo Umberto , De Amicis Francesca , Barone Ines , Giordano Cinzia , Malivindi Rocco , Catalano Stefania , Andò Sebastiano
Published: April 2020

Introduction

Glioblastoma multiforme (GBM) is the most malignant form of glioma causing 3–4% of all cancer‐related deaths. Specific cancer treatment for GBM includes surgery, radiotherapy and chemotherapy with temozolomide. Despite the continuous development of new clinical therapies, the prognosis and survival of GBM patients remain dismal. Thus, the deregulated growth, the migratory and invasive capabilities of glioma cells remain the most important obstacles to the development of effective GBM treatments. Angiotensin II (Ang II)/Ang II receptor 1 (AGTR1) axis is expressed in normal and tumoral tissues, including human GBM. It has been reported in different experimental models that Ang II is able to stimulate aromatase expression through the AGTR1 activation. Different studies have demonstrated that aromatase enzyme is expressed in GBM cells and local estrogen production may act as an autocrine or paracrine factor in enhancing glioblastoma growth and progression. In the present study, we investigated if Ang II/AGTR1 signalling may modulate aromatase expression and consequently local estrogen production, thus influencing glioblastoma survival and growth.

Methods

The Aromatase expression role under treatment with Ang II has been assessed by RT‐PCR, Western blotting, Aromatase activity assay, Transient transfection assays, ELISA assay, cell proliferation assay (Thymidine incorporation), Boyden chamber migration and invasion assays, in the GBM cell line U‐87 MG.

Results

We identified in U‐87 MG cells that the Ang II up‐regulates the aromatase expression in terms of mRNA, protein levels, and its enzymatic activity. In order to evaluate if Ang II may activate aromatase at transcriptional level we transiently transfected U‐87 MG cells with a vector containing human aromatase promoter I.4, broadly distributed around the brain and widely expressed in glioblastoma cells. Ang II treatment induces an increased aromatase promoter transcriptional activity. Mutagenesis studies and chromatin immunoprecipitation analysis reveal that the integrity of specificity protein 1 (SP1) and interferon‐gamma activation site (GAS) binding sites, within the aromatase promoter is required for Ang II modulation of human aromatase I.4 promoter activity in U87‐MG cells. The enhanced aromatase activity was paralleled by an higher estradiol production concomitant with an increase in cell proliferation, migration and invasion in U‐87 MG cells. These effects are reversed by treatment with an AGTR1 antagonist and/or an aromatase inhibitor, such as Losartan and Anastrozole respectively.

Conclusions

Ang II/AGTR1 signalling through Aromatase enhances local estrogen production which in turn promotes tumor growth and invasiveness. Therefore, their combined inhibitory targeting may prospectively provide a novel therapeutic strategy for Glioblastoma treatment.
Heterogeneous distribution of ion transporters in enteroids enriched in different intestinal epithelial cell types
Authors: Pearce Sarah C. , Kishida Kunihiro , Al-Jawadi Arwa , Shiarella Reilly J. , Auvinen Madelyn J. , Flores Juan A. , Yu Shiyan , Gao Nan A. , Ferraris Ronaldo P.
Published: April 2020

The small intestine which is characterized by different cell types lining specific sites along the crypt‐villus axis plays a critical role in water, electrolyte and mineral balance. Normally, net fluid absorption by the small intestine occurs mainly because ion‐dependent fluid absorption in the villus column is greater than ion‐driven fluid secretion in the crypt. To generate these site‐specific ion gradients, appropriate transporters must be strategically located in cells lining the crypt or villus regions. The multivalent ions calcium and iron also function as essential minerals, and deficiency in these minerals is highly prevalent worldwide. The crypt‐villus site of many electrolyte and mineral transporters has been localized by immunocytochemistry, but their expression in rare cell types like stem cells (ISC) is unknown. To test the hypothesis that representative ion and mineral transporters are distributed heterogeneously among the various intestinal cell types, we made enteroids enriched in ISC, enterocytes (ENT), goblet (GOB), and Paneth (PAN) cells. We then confirmed by cell‐type specific biomarkers that ISC, ENT, GOB and PAN enteroids were enriched in stem cells (~90% of all cells per organoid), enterocytes (90%), goblet (80%) and Paneth (65%) cells, respectively. ENT and GOB enteroids had the highest mRNA levels of the chloride‐anion exchanger DRA and sodium‐hydrogen exchanger NHE3, while the sodium‐bicarbonate cotransporter NBCe1 was expressed primarily in ENT enteroids. Conversely, the chloride channel CFTR and sodium‐potassium‐chloride cotransporter NKCC1 mRNA were mainly expressed in ISC and PAN enteroids. Findings confirm previous immunocytochemical localization of NBCe1 and DRA in the villus region lined by enterocytes, with CFTR and NKCC1 mainly in intestinal crypts containing Paneth and stem cells. Basolateral NKCC1 staining was observed in ISC and PAN, but not in ENT and GOB, enteroids. The membrane location and expression of NKCC1 in crypt‐dwelling cells reflect its prominent role in intestinal fluid secretion. Members of the intestinal epithelial transport system for iron (DMT1, FTL, IREG1 and HEPH) showed homogeneous distribution in all cell types. The calcium transporters TRPV6 and PMCA1 were expressed least in ENT and GOB, respectively. Our findings depict a mostly heterogeneous distribution of ion transporters in different cell types, reflecting their likely distribution along the crypt villus axis in vivo, suggesting that appropriate expression in different cell types may be essential for ion and mineral homeostasis.

Support or Funding Information

Supported by NSF 1456673, 1754783 (RF) and NIH R01AT010243 (RF, NG)
Inhibition of colorectal tumor formation using a new miR‐210 therapeutic inhibitor that targets the Lnc RNA Xist and Nme1
Authors: Eliason Steven L. , Hong Liu , Cao Huojun , Sweat Yan , Chalkley Camille , Liu Qi , Qi Hank , Xu Hongwei , Zhan Fenghuang , Amendt Brad A.
Published: April 2020

In this report we investigate the role of microRNA‐210 (miR‐210) in the etiology of colorectal cancer (CRC) using both cell‐based and xenograft mouse models. We show that miR‐210 expression is higher in the colon cancer cell line SW620 versus normal colon epithelium and has been associated with decreased cancer survival rates. Stable expression of our new miR‐210 inhibitor changes the morphology, proliferation and gene expression of SW620 cells and tumor growth. RNAseq identified potential targets of miR‐210 and qPCR has verified several of these targets. We find that inhibition of miR‐210 can act as a tumor suppressor and reduce tumor formation of SW620 colon cancer cells in xenograft mouse models. Direct comparison with capecitabine treatments demonstrates the effectiveness of the miR‐210 inhibitor treatment. The direct administration of the miR‐210 inhibitor (MiRIS) to existing tumors can inhibit tumor growth in both NSG and Foxnu/j mouse models. The inhibition of miR‐210 can cause alterations in hypoxia responses and upregulation of several tumor suppressor genes, including Nme1. We demonstrate that the long non‐coding transcript XIST is regulated by miR‐210 and XIST expression is decreased in colon tumors. We show that XIST expression correlates with the expression of the tumor suppressor gene Nme1. Furthermore, the inhibition of miR‐210 effects epigenetic marks of the NME1 loci, with increases in H3K4me3 and H3K27ac near the transcription start site (TSS). We show that our new miR‐210 inhibitor can be used as an effective colorectal cancer therapeutic tool.
Chronic Binge Alcohol and Gonadal Hormone Loss Mediate Adipose Tissue Metabolic Maladaptations
Authors: Poret Jonquil Marie , Simon Liz , Molina Patricia
Published: April 2020

Adipose tissue maladaptation is central to metabolic alterations in HIV, unhealthy alcohol use and obesity. This involves inflammation, adipokine disruption and dysfunction of adipocytes and the adipocyte extracellular milieu. HIV and simian immunodeficiency virus (SIV) have been shown to increase adipose tissue extracellular matrix (ECM) collagen deposition and impair adipose derived stem cell (ADSC) differentiation. Several aspects of adipocyte biology are regulated by ECM structure and composition. All proteins, regulators and secreted factors contained within the ECM are collectively called the matrisome and largely determines tissue stiffness. In several organs, tissue stiffness has been shown to disrupt normal cellular function and development, but little is known about tissue stiffness in adipose tissue. Using a relevant preclinical model of HIV‐infection, the objective of our study was to investigate the differential effects of chronic binge alcohol (CBA) administration and ovarian hormone loss on alterations in omental (OmAT) and subcutaneous (SAT) adipose tissue phenotype. CBA or isovolumetric water (VEH) was administered through an intragastric catheter for 30 minutes, 5 days a week, with blood alcohol concentrations reaching 50–60 mM. Three months after initiation of CBA administration, macaques were inoculated with SIVMac251 and 2.5 months later initiated on ART. After 1 month of ART, macaques underwent sham surgery or ovariectomy. Our preliminary data indicates that CBA administration increases viral load in adipose tissue. Depot differences were also detected between SAT and OmAT. OmAT has smaller adipocytes compared to SAT with higher anti‐fibrotic gene expression. SAT has higher collagen picrosirius red staining compared to OmAT with higher hormone sensitive lipase lipolytic gene expression. Ovariectomy produced similar effects in both adipose depots by decreasing adipocyte cell size and fibrotic gene expression. Taken together, preliminary data suggest that the maladaptive changes due to alcohol and gonadal hormone loss is potentially depot specific and is mediated by changes of the extracellular matrix. The contributing role of extracellular alterations on adipocyte function and a high fat diet to exacerbate adipose tissue maladaptation are investigated to identify adipose specific mechanisms that can be targeted to improve metabolic dysregulation.

Support or Funding Information

NIH/NIAAA P60 AA009803 and 5T32AA007577‐19
XLαs is a novel activator of PLCβ4
Authors: Phan Hoa T.N. , He Qing , Bastepe Murat , Smrcka Alan V.
Published: April 2020

Phospholipase C (PLC) enzymes are essential for normal cellular function. Upon stimulation by GPCRs or RTKs, PLCs hydrolyze phosphatidylinositol 4,5‐bisphosphate (PIP2) at cell membranes to generate diacylglycerol (DAG) and inositol 1,4,5‐trisphosphate (IP3). DAG and IP3 are important second messengers in signal transduction. DAG regulates activity of protein kinase C and IP3 mobilizes intracellular Ca2+, which initiate multiple signaling cascades and cellular processes. Dysregulation of PLC function gives rise to pathological processes. Among six mammalian PLC subfamilies, PLCb isozymes are activated by Gaq, Gbg, small G proteins and thus, play significant roles in various cellular activities.

Extra‐large stimulatory Gα (XLαs) is a large variant of Gαs. Biochemically, XLαs activation by GPCR mediates cAMP‐ generation similarly to Gαs, however, Gαs and XLαs ‐‐‐have distinct cellular and physiological functions. He et al. suggests that XLαs is able to stimulate IP3 signaling in vivo. In this study, we aim to identify the PLC isozyme that mediates this phenomenon. By co‐expressing XLαs with different PLC constructs in cells and measuring total IP production, we identify that XLαs stimulates PLCβ4 to generate IP3. This activity is specific to XLαs because wild‐type and constitutively active Gαs do not activate PLCβ4. The G‐protein domain and membrane anchor of XLαs are required for its ability to stimulate PLCβ4. Our data also show that XLαs‐mediated PLCβ4 activation is PKA‐ and EPAC‐independent. Further studies are underway to determine whether this is a direct binding and activation event, and the role of XLαs stimulation of PLCb4 in physiological systems.

Overall, we show that XLαs can stimulate PLCβ4 in cell and this novel signaling axis potentially play important roles physiologically.

Support or Funding Information

This work is funded by the NIH 5R35GM127303 (to AVS) and the Upjohn postdoctoral fellowship (to HTNP).
Cognitive function and regional body water analysis across the lifespan
Authors: Lee Jinhyun , Dudley-Javoroski Shauna A. , Dubey Olga , McCue Patrick M. , Johnson Kristin A. , Petrie Michael Arlyn , Shields Richard K.
Published: April 2020

Objective

Cognition decline occurs with age and adversely influences quality of life. Recent work suggests that systemic inflammation increases with age and may be associated with homeostasis of body water compartment levels. The extent to which fluid cognition is influenced by age and body water compartment distribution has not been carefully explored. Bioimpedance algorithms now enable us to analyze regional anatomical body water and determine estimates of the intra‐cellular and extra‐cellular water content levels. The ratio of the extra‐cellular water (ECW) relative to the intra‐cellular water (ICW) may provide a non‐invasive method to estimate systemic inflammation and predict cognitive function. In this study, we aspired to understand how age and water content predict fluid cognitive function in humans.

Purpose

We determined the correlation between anatomical regions of the body for water content (ECW/ICW) and cognitive function as a function of age.

Methods

There were 101 participants (Male: 50, age<40: 62, age>40: 39, age mean:39.9±18.7). We used a bioimpedance analysis device (InBody S10), which can estimate six regional extracellular water (ECW) and intracellular water (ICW) readings. We calculate the ratio of ECW to ICW (ECW/ICW) for each of the six regions: left leg, left arm, right leg, right arm, trunk, and total. The NIH cognition toolbox Pattern Comparison Processing Speed (PCPS), Picture Sequence Memory (PSM), Dimensional Change Card Sort (DCCS), FLANKER inhibitory control and attention (FLANKER) and List Sorting Working memory (LSW) were used to test cognitive function. The composite score was calculated from these five tests.

Results

The ECW/ICW of the left leg was significantly associated with all cognitive test results in the cohort with R‐squared values of 0.3, 0.46, 0.06, 0.12, 0.39, and 0.4 for FLANKER, LSW, PSM, PCPS, and the composite score, respectively. Age was highly correlated with cognitive test results in the cohort with R‐squared values of 0.32, 0.45, 0.07, 0.27, 0.5, 0.53, for the FLANKER, LSW, PSM, PCPS, and composite, respectively. FLANKER was highly correlated with bioimpedance indices in the older people with R‐squared values of 0.24, 0.22, 0.24, and 0.25 for the ECW/ICW total, ECW/ICW trunk, ECW/ICW right leg, and ECW/ICW left leg, respectively. FLANKER was not correlated with age in the older people with R‐squared value of 0.05.

Conclusions

The associations between cognitive function and bioimpedance indices were similar or higher than the associations between cognitive function and age. Future studies are needed to verify that ECW/ICW is associated with systemic inflammation and is a rigorous predictor of cognitive change, at any age.

Support or Funding Information

Research Support

This study was funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development: R01HD084645, R01HD082109
Effects of acute smoking on heart rate variability in students
Authors: VOLKOVA IRINA , DIMITRIEV DMITRY , SAPEROVA ELENA , FILIPPOVA IRINA
Published: April 2020

Cigarette smoking is a major cause of preventable morbidity and mortality in the world. There is altered autonomic activity in smokers which also predisposes to cardiovascular morbidity and mortality. To monitor the autonomic activity, heart rate variability has emerged as efficient tool. This study attempted to clarify the acute effects of cigarette smoking on autonomic nervous function among smokers.

Twenty one light smokers (smoking less than 10 cigarettes a day) with a mean age of 22.36±0.16 years take part in the study (52% men, 48% women). They were studied in the morning hours in a quiet room at a comfortable temperature. Time domain (SDNN, RMSSD, pNN50), frequency domain (TP, HF, VLF, LF and LF/HF ratio) and nonlinear (SD1, SD2, ApEn, SampEn, D2, DFA1, DFA2) parameters of HRV were obtained from all participants for 10 minutes before and 10 minutes after smoking a cigarette while subjects were resting in a supine position and. Statistical analysis was performed using Mann‐Whitney test.

There were no significant differences in heart rate during non‐smoking and smoking period (70.75±2.30 vs 73.37±2.33 beat/min, p=0.217). Time domain parameters were found to be lower during smoking period than that during non‐smoking period (SDNN: 38.07±3.91 vs 50.53±5.64 ms, p=0.001; RMSSD: 39.39±4.87 vs 56.4±7.57 ms, p=0.001 and pNN50: 17.08±3.61 vs 28.63±4.28 %, p=0.002). Frequency domain measures were also significantly lower during smoking period than that during non‐smoking period (TP: 1578.31±387.76 vs 2975.75±788.91 ms2, p=0.001; VLF: 52.52±10.11 vs 108.96±24.56 ms2, p=0.008; LF: 719.5±130.54 vs 1210.54±262.29 ms2, p=0.013 and HF: 804.96±275.86 vs 1650.12±578.23 ms2, p=0.0009). Nonlinear measures were significantly lower during smoking period than that during non‐smoking period (SD1: 27.88±3.45 vs 39.92±5.36 ms, p=0.001; SD2: 45.79±4.45 vs 58.84±6.5 ms, p=0.003; D2: 1.83±0.36 vs 3.09±0.37, p=0.013). The other HRV parameters including LF/HF ratio, pLF, pHF, ApEn, SampEn, DFA1, DFA2 were not significantly different.

These results suggest that acute smoking has been associated with increased activity of the sympathetic nervous system and decrease in vagal cardiac control.
The Utility of White‐Light 3D Scanning in the Cadaver Lab: Examples Applicable to Iliac Crest Grafting for Mandibular Reconstruction
Authors: Hobeika Natalie A. , Bolden Alexys M. , Burkett Jesse T. , Shockley J. Mason , Zdilla Matthew J. , Lambert H. Wayne
Published: April 2020

Performing imaging studies such as CT and MRI on cadavers in a gross anatomy setting may be particularly challenging or not feasible. Therefore, other imaging modalities should be investigated. This report highlights the utility of white‐light 3D scanning in a gross anatomical setting as applied to a shape compatibility analysis among iliac crest grafts and native mandibular anatomy in seven cadavers. Upon resection of iliac crests and mandibles, white‐light 3D scanning was performed, digital meshes (e.g., .ply files) were edited, and geometric morphometric analysis was performed using varied software to determine regions of shape compatibility/incompatibility. The results of the white‐light scanning resemble the detail of 3D reconstructions from CT and MRI for surface renderings; though, white‐light scanning has limitations with regard to visualizing underlying structures. The study concludes that white‐light scanning has utility in the gross anatomy setting, particularly as it related to 3D surface analysis as demonstrated by the analysis of the iliac crest graft as it relates to the anatomy of the mandible. Also, digital models can be manipulated with software and also be printed to produce physical models. White‐light scanning is suited for the gross anatomy laboratory setting and provides highly accurate digital renderings that are applicable to research as well as basic science and clinical education.

Support or Funding Information

WVU INTRO (Initiation to Research Opportunities) Summer Research program; WV Research Challenge Fund [HEPC.dsr.17.06]
Maternal and postnatal diet play important roles on cardiometabolic health of offspring
Authors: Merlin Andrea Chiñas , Gonzalez Kassandra , Lota Jake , Chicco Adam J. , Chung Eunhee
Published: April 2020

The prevalence of obesity and type 2 diabetes mellitus (T2DM) among children is alarmingly increasing in the United States, and children born from obese mothers are at greater risk for diabetes and cardiovascular disease. Cardiac mitochondrial dysfunction may contribute to heart disease associated with T2DM and obesity. The purpose of this study was to test hypothesis that a maternal obesogenic diet would alter offspring cardiac mitochondrial function and systemic metabolism. Female CD‐1 mice were fed either a high‐fat, high‐sucrose diet (HFHS) or a refined low‐fat, low‐sucrose diet (LFLS) for eight weeks before initiation of pregnancy, gestation, and during lactation. At 21 days, male mice were randomly divided into 3 groups: offspring of HFHS dams weaned onto a standard chow diet (HC), HFHS diet (HH), or offspring of LFLS dams continue fed to LFLS diet (LL). Glucose and insulin tolerance tests were performed in 6 month‐old male offspring, followed by sacrifice for evaluation of cardiac mitochondrial respiratory function. HFHS diet‐induced maternal obesity impaired glucose tolerance and insulin sensitivity in HH offspring. Switching offspring to the control diet (HC) did not decrease body weight associated with maternal obesity, but significantly decreased subcutaneous fat mass compared to the HH, and preserved glucose tolerance and insulin responsiveness to near LL levels. In freshly isolated cardiac mitochondria, maximal oxidative phosphorylation (OXPHOS)‐linked respiratory capacity and coupling efficiency was significantly higher in the HC in the presence of fatty acid substrate (palmitoylcarnitine) compared to LL. In the absence of fatty acids, OXPHOS‐linked respiratory capacity and OXPHOS coupling control were not altered. These results indicate that both maternal and postnatal diet modulate offspring cardiac mitochondrial metabolism, favoring greater fatty acid oxidation in offspring fed a control diet following maternal obesity.

Support or Funding Information

This study was funded by the American Physiological Society Research Career Enhancement Award and the University of Texas at San Antonio, Office of the Vice President for Research and the College of Education and Human Developments.
Angiotensin II Type I Receptor Agonistic Autoantibody Blockade Improves Cerebral Blood Flow Autoregulation, Blood Brain Barrier Permeability, and Hypertension in the Pre‐Clinical Rat Model of Preeclampsia
Authors: Cunningham Mark W. , Duncan Jeremy W. , Azubuike Daniel , Booz George , Fisher Brandon , Williams Jan , LaMarca Babbette
Published: April 2020

Background

Preeclampsia (PE) is a pregnancy disorder that is characterized by an increase in blood pressure, angiotensin II type1 receptor agonistic autoantibodies (AT1‐AA), and neurological complications. The pre‐clinical RUPP (reduced uterine perfusion pressure) rat model of PE displays many of the clinical pathologies associated with PE, such as increased blood pressure, AT1‐AAs, blood brain barrier (BBB) permeability, and impaired cerebral blood flow (CBF) autoregulation.

Hypothesis

We hypothesized that specific inhibition of AT1‐AA, using the epitope binding 7 amino acid peptide sequence (7AA), will improve blood pressure, BBB integrity, and CBF autoregulation in RUPP rats.

Methods

Sprague Dawley rats, were divided into 3 groups: normal pregnant (NP) (n=19), RUPP (n=19), and RUPP+AT1‐AA inhibition (7AA) (n=21). RUPP surgery was performed on gestational day (GD) 14 and 7AA (2μg/μl) was administered via osmotic minipumps. GD 19, mean arterial pressure (MAP) was determined and BBB permeability was assessed using the in‐vivo imaging system (IVIS). CBF was measured by laser Doppler flowmetry through a 4 mm x 4 mm cranial window under anesthesia. MAP was elevated step‐wise from 100–190 mmHg by infusion of phenylephrine to determine changes in CBF.

Results

MAP was increased in RUPP vs NP (124±3 vs 100±1 mmHg, p<0.05) and decreased in RUPP+7AA vs RUPP (105±2 vs 124±3 mmHg, p<0.05) rats. Using IVIS, Texas Red/FITC green fluorescent imaging was decreased in RUPP+7AA vs RUPP (0.2±0.001 vs 0.6±0.001, p<0.05), indicating improvement in BBB permeability with AT1‐AA inhibition. CBF autoregulation was impaired in RUPP vs NP rats at 140 mmHg (142±7 vs 119±4%, p<0.05) and 160 mmHg (157±10 vs 129±6%, p<0.05) (both pressures are within the normal CBF autoregulatory range). AT1‐AA inhibition prevented impaired CBF at 140mmHg (125±5 RUPP+7AA vs 142±7% RUPP, p<0.05) and 160 mmHg (125±9 RUPP+7AA vs 157±10% RUPP, p<0.05). Furthermore, the increases in CBF at pressures above the autoregulatory range, 180 mmHg (122±10 vs 198±17%, p<0.05) and 190 mmHg (123±11 vs 208±19%, p<0.05), was prevented in RUPP+7AA vs RUPP, respectively.

Conclusion

AT1‐AA inhibition improved blood pressure, BBB permeability, and CBF autoregulation in the pre‐clinical RUPP rat model of PE, indicating that AT1‐AA inhibition may be a potential therapy used to improve neurological complications during PE.

Support or Funding Information

Supported: AHA18CDA34110264