bluuebunny/crossref_metadata_2025 huggingface dataset:
Microengineered hydrogels for tissue engineering (82.4)
Authors: Khademhosseini Ali
Published: June 2021

Microengineered hydrogels for stem cell bioengineering and tissue regeneration Micro‐ and nanoscale technologies are emerging as powerful tools for controlling the interaction between cells and their surroundings for biological studies, tissue engineering, and cell‐based screening. In addition, hydrogel biomaterials have been increasingly used in various tissue engineering applications since they provide cells with a hydrated 3D microenvironment that mimics the native extracellular matrix. In our lab we have developed approaches to merge microscale techniques with hydrogel biomaterials for directing stem cell differentiation and generating complex 3D tissues. In this talk, I will outline our work in controlling the cell‐microenvironment interactions by using patterned hydrogels to direct the differentiation of stem cells. In addition, I will describe the fabrication and the use of microscale hydrogels for tissue engineering by using a ‘bottom‐up’ and a ‘top‐down’ approach. Top‐down approaches for fabricating complex engineered tissues involve the use of miniaturization techniques to control cell‐cell interactions or to recreate biomimetic microvascular networks within mesoscale hydrogels. Our group has also pioneered bottom‐up approaches to generate tissues by the assembly of shape‐controlled cell‐laden microgels (i.e. tissue building blocks), that resemble functional tissue units.
Proteomic analysis of changes in protein expression in rat liver by oral administration of isoleucine (820.3)
Authors: Yoshizawa Fumiaki , Koike Shinichiro , Sugahara Kunio
Published: June 2021

The branched chain amino acids (BCAAs) have recently been recognized as having functions other than simple nutrition. Among the BCAAs, isoleucine acts as a signal in glucose metabolism. Isoleucine depresses gluconeogenesis in the liver, in addition to stimulating both glucose uptake in skeletal muscle and whole body glucose oxidation, thereby leading to a hypoglycemic effect in rats. To further evaluate the characteristics of isoleucine as a nutritional signal, we performed proteomic analysis on liver proteins. Eighteen hours fasted male Wistar rats were divided into 3 groups. One group of animals was sacrificed without any treatment as a control. Remaining two groups of animals were orally administered 135mg/100g body weight isoleucine and then sacrificed at 1h or 3h after administration. There were 4 spots that were changed at 3h after administration of isoleucine, and 3 spots of 4 were identified as mitochondrial aldehyde dehydrogenase (ALDH2), alpha‐enolase (ENO‐1) and pyruvate dehydrogenase phosphatase regulatory subunit (PDPr). The changes in the spots of ALDH2 and ENO‐1 were caused by phosphorylation modification. ENO‐1 was phosphorylated and thus inactivated by isoleucine administration. PDPr expression was 1.5‐fold greater than that of control. PDPr has negative effects on the activity of PDP, which activates pyruvate dehydrogenase (PDH) by dephosphorylation. These indicate that isoleucine administration may lead to inhibit glycolysis by decreasing the activity of ENO‐1 or by inhibiting the activity of PDH through inactivation of PDP. Based on these results, we speculate that isoleucine acts as a signal, which regulates not only gluconeogenesis but also glycolysis in the liver.
Effects of dairy ingredients on cytokine responses to LPS challenge in mice (822.5)
Authors: Price Tara , Baskaran Sangeetha , Walzem Rosemary
Published: June 2021

Gut permeability to bacterial cell wall components, e.g.LPS, are thought to play a causative role in chronic systemic inflammation leading to disease. The effects of individual protein sources, namely isolated soy protein (ISP), dried whole milk powder (DWMP), milk fat globule membrane (MFGM) or milk protein concentrate (MPC) on cytokine responses to LPS (Salmonella) challenge was studied using whole blood from C57BL/6J mice (n=10/group) following 13 wk of test diet feeding. Isocaloric and isonitrogenous diets provided 20 EN% protein, 50 EN% carbohydrate and 30 EN% fat. Mice grew and consumed feed similarly. Multiplex assay for basal inflammatory cytokines showed lowest values for DWMP in all cases and significant for IL‐1β and TNF‐α (p<0.05). No differences in basal IL‐10 were observed. In response to LPS challenge IL‐12(p70), IFN‐γ, TNF‐α and MIP‐2 were unchanged. Fold increases in IL‐1β and IL‐6 were lowest for MPC while ISP and DWMP showed the greatest increase in IL‐1β and IL‐6, respectively (p<0.05). IL‐10 was similar in MFGM and MPC but MFGM was significantly higher than ISP and DWMP after LPS challenge. Dairy protein components appear to differentially influence cytokine response to LPS. Intact dairy protein (DWMP), appears least inflammatory in a normal gut, while specific milk protein fractions (MPC and MFGM) allow for an improved and more highly regulated immune response to LPS challenge.

Grant Funding Source: National Dairy Council Project #1120 administered by Dairy Research Institute
Defining the role of pulse grains in cancer prevention (824.10)
Authors: Thompson Henry , McGinley John , Echeverria Dimas , Brick Mark
Published: June 2021

Pulse crops, including dry beans, dry peas, chickpeas, and lentils, are grain legumes that serve as staple food crops for populations around the world. Since ancient times, pulses have been combined with cereal grains, primarily wheat, rice, or corn, or with tubers such as potato or cassava to provide a balanced source of protein and carbohydrate and a nutritionally adequate diet when combined with seed oils, fruit and vegetables. In this setting, the established ratio of cereal grains to pulses to meet nutritional requirements is 2:1; however, over the last 60 years both production and consumption patterns, globally, have changed to 8:1. One consequence of these changes has been a marked but largely unappreciated exclusion of pulse crops from the diet. In this study, the effects of one type of pulse crop, common bean, was evaluated in a dual model for experimentally induced colon and breast cancer in female Sprague Dawley rats injected with both 1‐methyl‐1‐nitrosourea and azoxymethane. Specific varieties of cooked, freeze‐dried bean were incorporated into a purified diet and fed during the post initiation phase of the disease process. Depending on common bean variety, the occurrence of both aberrant crypt foci in the colon and mammary carcinomas were inhibited when beans were included in the diet compared to the control without bean. Magnitudes of inhibition ranged from 25 to 50%. Potential contributions of various components of cooked bean are considered including content of fiber and various phytochemicals. The lack of significant amounts of common bean and other pulse grains in the US diet may represent an underexploited public health opportunity especially in the context of re‐establishing complementary patterns of consuming pulse grains and cereal crops.

Grant Funding Source: PHS R01‐ CA172375
Hsp90 inhibition abolishes LPS‐mediated NF‐κB transcriptional activation independent of IKKβ in human lung microvascular endothelial cells (HLMVEC) (847.7)
Authors: Thangjam Gagan , Joshi Atul , Barabutis Nektarios , Charalampos Birmpas , Patel Vijay , Fulton David , Catravas John
Published: June 2021

LPS promotes NF‐κB‐dependent inflammation by activating IKKβ which phosphorylates inhibitory IKB proteins leading to their degradation. Basally, NF‐κB exists in an inactive cytoplasmic complex with IKB. Stimulus‐dependent degradation of IKB allows NF‐κB to translocate into the nucleus and initiate transcription. Hsp90 is essential for the activation of a number of kinases, including IKKβ, and thus an important regulator of the NF‐κB pathway. Here we demonstrate that hsp90 inhibition by 17‐AAG attenuated LPS‐induced NF‐κB reporter and target gene activation in HLMVEC. Similarly, inhibition of upstream TAK‐1 and IKKβ blocked IKBα phosphorylation/degradation by LPS. However, 17‐AAG blocked the LPS‐induced phosphorylation/ubiquitination of IKBα without preventing its degradation. Indeed, in the presence of 17‐AAG, LPS degraded a phosphorylation/ubiquitination‐deficient mutant IKBα. This 17‐AAG‐mediated degradation did not involve IKKβ, since addition of excess IKKβ inhibitor, after 17‐AAG, failed to recover IKBα protein levels. Instead, 17‐AAG prevented the LPS‐induced recruitment of NF‐κB and its co‐activator CBP to NF‐κB target gene promoter. This was associated with the prevention of LPS‐induced core histone H3 eviction and N‐terminal phosphorylation of RNA polymerase II. Thus, we conclude that hsp90 inhibition does not affect IKKβ‐dependent NF‐κB signaling but blocks NF‐κB transcriptional activation via an epigenetic mechanism in HLMVEC.

Grant Funding Source: HL093460
Cocaine‐induced locomotor sensitization persists in rats that escalate cocaine self‐administration in a short‐access procedure (848.6)
Authors: Mandt Bruce , Copenhagen Leland , Zahniser Nancy , Allen Richard
Published: June 2021

The role of sensitization in cocaine addiction remains debatable. In rats, non‐contingent cocaine‐induced locomotor sensitization is well documented. However, the relationship between this measure and measures of cocaine self‐administration is less clear. For example, repeated non‐contingent cocaine pre‐exposure enhances acquisition of self‐administration and increases break points (BPs) for cocaine. Following escalation of intake or BPs, however, some studies report tolerance to cocaine’s locomotor‐stimulating effects. Importantly, sensitization is often assessed after long self‐administration histories or high levels of cocaine exposure. We have recently developed a method for assessing escalation early in self‐administration. Using this method, here we report on the relationship between cocaine‐induced locomotor sensitization and escalation early in the drug‐taking process. Cocaine‐induced (10 mg/kg, i.p.) locomotor activity was assessed on 3 occasions: 1) in a drug‐naïve state, 2) after 5 days of non‐contingent cocaine administered in operant chambers, and 3) 48 hrs after the 10th active self‐administration session. Once‐daily non‐contingent cocaine administration (0.8 mg/kg, i.v.) in the self‐administration context induced robust locomotor sensitization. When allowed to self‐administer cocaine (0.8 mg/kg, i.v.), rats rapidly escalated intake over 10 post‐acquisition sessions. Notably, cocaine‐induced locomotor sensitization was preserved following self‐administration, despite escalation. These data suggest cocaine‐induced locomotor sensitization and escalation are not mutually exclusive, but rather, may occur in tandem during the earliest stages of drug taking.

Grant Funding Source: Supported by NIH Grant R01 DA004216
Estrogen signaling alterations in resistance arteries postpartum (849.6)
Authors: Royal Crista , White Richard
Published: June 2021

Few studies have examined the after‐effects of childbirth on the female vasculature – especially the resistance microvasculature of non‐reproductive tissues, which are a major determinant of blood pressure. 17β‐estradiol (E2), an important vasoactive hormone, normally declines after parturition; however, many women artificially increase estrogen levels after pregnancy by taking oral contraceptives.

Our hypothesis was that parturition attenuates E2‐induced relaxation in mesenteric arteries by decreasing nNOS expression. We measured a 73% decrease in serum E2 levels in the postpartum state compared to nulliparous Sprague‐Dawley rats. We found that microvessels from postpartum animals exhibited: 1) endothelium‐independent, E2‐induced relaxation (intact 18.78 ± 3.84% vs denuded 15.37 ± 3.30%), 2) impaired E2‐induced relaxation due to decreased NOS activity (L‐NMMA, ‐17.54 ± 15.66% vs postpartum controls 12.98 ± 2.94% relaxation at 1000nM E2), 3) impaired nNOS‐dependent, E2‐induced relaxation, and 4) decreased nNOS expression (down 39% p=0.0077). Because E2 has been shown to increase nNOS protein expression, we propose that lower E2 levels after parturition decrease expression of nNOS, leading to a reduced vasodilatory capacity of resistance microvessels.

Grant Funding Source: Supported in part by NIH HL073890
Transport of high density lipoproteins though endothelial cells (855.7)
Authors: Perisa Damir , Käch Andres , von Eckardstein Arnold , Rohrer Lucia
Published: June 2021

Atherosclerosis is a progressive accumulation of lipids in the arterial intima leading to plaque formation. Epidemiological studies show an inverse association of high density lipoprotein (HDL) cholesterol with cardiovascular events. HDL has multiple anti‐atherogenic functions, some of which take place in the vessel wall. To get access to the intima and the lipid‐laden macrophages, HDL has to pass the endothelial barrier.

To elucidate the itinerary of HDL through endothelial cells (ECs), we investigated localisation and distribution of fluorescently and 1.4 nm Nanogold labelled HDL in fluorescent (FL) as well as electron microscopy (EM). For EM, we used high pressure freezing (HPF) and to avoid lipid extraction we developed a fast method for silver enhancement of the nanogold‐HDL after fixation in OsO4.

HDL is detectable in ECs in a concentration and time dependent manner. Colocalisations of HDL with dextran were negative excluding fluid phase uptake and transferrin and albumin show only slight colocalisation indicating an atypical uptake mechanism.

Further experiments showed that HDL is neither targeted to lysosomes nor to the Golgi or the endoplasmatic reticulum. Only small amounts can be detected in early endosomes (Rab5) and endosome to trans‐golgi network (Rab9), but not at all with late endosomes (Rab7), the recycling endosomes (Rab11a) or vesicles involved in trans‐Golgi network sorting (Stx6). EM ultrastructure shows HDL to be located mainly in multivesicular bodies.

We corroborated our findings in aortic artery endothelium. Live microscopy experiments as well as biochemical data help us to understand the itinerary of HDL through aortic ECs.
Phenotypic differences in putative EPCs based on in vitro cultivation (855.8)
Authors: Rocha Natalia , Bammert Tyler , Beckstrom Collin , Kavlich Philip , Lincenberg Grace , Greiner Jared , DeSouza Christopher
Published: June 2021

Circulating endothelial progenitor cells (EPCs) have been extensively studied over the past decade. Isolation techniques have differed in the literature based on whether the non‐adherent or adherent cells from fibronectin‐coated plates are studied. We determined whether there are phenotypic differences in cellular function between putative EPCs derived from the adherent or non‐adherent mononuclear cell population. Peripheral blood samples were collected from 15 healthy adults. Mononuclear cells were isolated by Ficoll density‐gradient centrifugation and plated on 6‐well plates coated with human fibronectin. After 2 days, non‐adherent cells were collected; in a separate culture, adherent cells were removed and collected after 7 days. Migratory capacity (Boyden chamber), angiogenic growth factor release (ELISA) and apoptosis (TUNEL) were assessed in both cell types. Migration to both VEGF (505+86 vs 254+ 55 AU) and SDF‐1 (498+83 vs 231+46 AU) were ~2‐fold higher (P<0.05) in the non‐adherent vs adherent cells. Release of angiogenic factors: granulocyte colony‐stimulating factor and hepatocyte growth factor were not different between cell types. Apoptotic response to staurosporine was significantly lower (~60%) in non‐adherent cells. In summary, non‐adherent and adherent cultured putative EPCs demonstrate functional phenotypic differences in migratory capacity and apoptotic susceptibility.
Endogenous kappa opioid receptors play a role in angiotensin II high salt diet hypertensive rats (860.24)
Authors: Nguyen Tin , Franklin Cynthia , Hollister Bernadette , Tang Jeffrey , Rangel Yolanda , Gottlieb Helmut
Published: June 2021

Central kappa opioid receptor (KOR) activation produces a marked increase in renal sympathetic nerve activity with a concurrent free water diuresis in conscious rats. However, the role of KOR in the angiotensin II high salt diet (AngII) mediated hypertension model is yet to be determined. This study examines cFOS expression in several brain regions of AngII rats as compared to normal rats. In addition, cFos expression in these brain regions was examined following intracerebroventricular (ICV) microinjection of norbinaltorphimine (Nor‐BNI; 1 μg/5μl), a selective KOR antagonist. Alternate sets of forebrain sections were processed for c‐Fos using a commercially available antibody. Sections were also double labeled with vasopressin to anatomically define specific brain regions. Fos expression was significantly increased in several lamina terminalis and hypothalamic sites. ICV nor‐BNI produced significant decreases in cFos staining in the PVN (AngII, 106 ± 12; nor‐BNI 62 ± 7; P < .01) and OVLT (AngII, 132 ± 7; nor‐BNI 73 ± 5; P < .05). There was no significant change in urine and sodium excretion in all groups. Altogether, this data provides new information concerning the role of KOR in the regulation of blood pressure

Grant Funding Source: 5SC2HL104639‐03
Characterization of a cGMP‐dependent protein kinase I gamma nuclear localization sequence (866.2)
Authors: Chen Jingsi , Roberts Jesse
Published: June 2021

PKGI regulates vascular smooth muscle cell (SMC) phenotype through incompletely understood mechanisms. Recently, we noted that cGMP stimulates PKGI proteolysis, in part by proprotein convertases, the nuclear localization of a constitutively active kinase fragment, PKGIγ, and transactivation of CRE‐dependent gene expression. PKGIγ appears to harbor an IL‐1α‐like NLS described by others (Gudi 1997). Here we tested whether this putative NLS determines PKGIγ compartmentation and function.

We observed that a single amino acid mutation in PKGIγ was sufficient to modulate its nuclear localization. Immuno‐localization studies revealed that rat aortic SMC transfected with plasmids encoding PKGIγ with a KIL(K‐>E)KRH mutation exhibited decreased nuclear PKGIγ compartmentation. Moreover, studies using a plasmid‐encoded nuclear phospho‐CREB S133 biosensor (Friedrich 2010) and fluorescence lifetime imaging revealed that these cells had less nuclear PKGI kinase activity even though immunoblotting revealed preserved cytosolic enzyme activity. Moreover, the nuclear function of PKGIγ was not due to cAMP signaling system cross talk as it persisted in cells treated with myr‐PKI, a PKA inhibitor.

These data indicate that a defined amino acid sequence regulates PKGIγ nuclear localization and function. Additional studies are warranted to test how this nuclear PKGIγ compartmentation regulates SMC phenotype.

Grant Funding Source: Supported by NIH grant R01HL096779
Exposure of human bronchial epithelial cells to fungal aeroallergens induces calcium mobilization and anion secretion by a mechanism involving oxidative stress (869.3)
Authors: O'Grady Kelly , Maniak Peter , Kita Hirohito
Published: June 2021

Aeroallergens produced by Alternaria alternata have been shown to induce life threatening exacerbations of airway inflammation in sensitized patients with severe asthma. Inflammation produced by Alternaria is associated with ATP release and purinergic receptor activation. In the present study the effects of Alternaria on signaling pathways and ion transport mechanisms associated with mucociliary clearance were investigated in an immortalized human bronchial epithelial cell line (16HBE14o‐ cells). Cells were grown on SnapwellTM membrane filters until a stable transepithelial resistance (>500 Ω) was established. Cell monolayers were then voltage clamped at zero mV and apically stimulated with 50 μM Alternaria extract to induce a time‐dependent increase in short circuit current (Isc) consistent with anion secretion. Treatment with the CFTR channel blocker, CFTRinh‐172 significantly reduced the Isc by ~25%. Furthermore, addition of the disulfonic stilbene compound DIDS (250 μM) abolished the Isc response. Measurements of the effect of Alternaria on intracellular Ca2+ concentration ([Ca2+]i) showed that the extract caused a persistent increase in [Ca2+]i that was blocked by chelating extracellular Ca2+ with EGTA. Pretreatment with 250 μM DIDS also abrogated the effects of Alternaria on [Ca2+]i but did not affect the increase in [Ca2+]i produced by 4 μM ionomycin. Similarly, pretreatment of monolayers with glutathione also blocked the Alternaria induced increase in [Ca2+]i. These results indicate that Alternaria aeroallergens are capable of stimulating CFTR and Ca2+‐dependent anion secretion. Moreover, the increase in [Ca2+]i appears to involve a DIDS‐sensitive uptake mechanism that is blocked when oxidant scavenger molecules are present in the extracellular solution.

Grant Funding Source: Supported by NIH: HL110539
Aeroallergens derived from Alternaria alternata evoke ATP release and chronic increases in intracellular Ca2+ in bronchial epithelial cells by a mechanism involving oxidative stress (869.4)
Authors: Patil Nandadevi , Maniak Peter , Kita Hirohito , O'Grady Scott
Published: June 2021

Alternaria aeroallergens have been previously shown to stimulate allergic inflammation and exacerbation of respiratory distress in patients with asthma. An early event following exposure to Alternaria extract is the sustained release of ATP and a persistent increase in intracellular [Ca2+] ([Ca2+]i) resulting from uptake of Ca2+ ions from the extracellular solution. In the present study we tested the hyopothesis that the release of ATP and the subsequent increase in [Ca2+]i was due to oxidative stress induced by exposure to Alternaria. ATP release following apical stimulation with Alternaria was measured in human primary bronchial epithelial cells using a luciferase/luciferin ATP measurement kit in combination with a modified luminometer that allows for tracking ATP release in real time. Pretreatment with glutathione (GSH) produced a concentration‐dependent inhibition of ATP release with an EC50 = 500 μM. Furthermore, GSH blocked the increase in [Ca2+]i typically observed following apical exposure to Alternaria. Moreover an increase in the expression of specific antioxidant protein mRNAs was also observed after 12 hours of treatment with Alternaria extract. These proteins included peroxiredoxins 1‐6, keratin 1, cyclooxygenase 2 (COX2) and oxidative stress responsive 1 protein with COX2 having the highest level of expression (~20 fold). These results implicate oxidative stress as an important initiating event in Alternaria evoked airway inflammation.

Grant Funding Source: Supported by NIH: HL110539
Therapeutic effects of mesenchymal stem cells on hepatocellular carcinma: tracking of cells using iron oxide nanoparticles (87.3)
Authors: Noorwali Abdulwahab , Faidaah Mamdooh , Atta Hazem , Damiati Laila , Filimban Najlaa , Al‐Grigry Mihal , Habib Hamid , Radwi Amer , Almarees Ali
Published: June 2021

Recently, a significant increase in the incidence of hepatocellular carcinoma (HCC) has been reported. However, early detection of the disease can help in selecting from various available therapies. Unfortunately, in advanced liver cancer cases, treatment options are very limited. In the present study, we point to the need to identify a new effective, less aggressive treatment approach. Advances in stem cell research, led us to consider cell‐based therapy for treating liver cancer.

It was previously reported that bone marrow derived mesenchymal stem cells (MSCs) have the tumor suppressive effects in an experimental HCC model in rats. In this work, we investigated the possible role of Wnt signaling in hepatic carcinogenesis and how it is influenced by by MSCs labeled with iron oxide nanoparticles.

Forty rats were used and were divided equally into four groups: a normal control group and 3 groups that received diethylnitroseamine and CCl4 to induce HCC. Then after induction, one group was treated with MSCs only, the second group with PBS (vehicle) only, and the third group with labeled MSCs with iron‐oxide nanoparticles. Gene expression of Wnt signaling target genes by reverse transcription‐polymerase chain reaction (RT‐PCR), in rat liver tissue, was measured. In addition, serum levels of liver function parameters and alpha fetoprotein were performed in all groups. Histopathological examination of the liver and organ samples from all groups was performed. Magnetic resonance imaging (MRI) was used to visualize MSCs loaded with iron oxide nanoparticles in the affected liver. We detected a significant tumor‐mass reduction in the group which received MSCs compared to the control groups.

The results of this work confirm the previous finding of a possible therapeutic effect of MSCs on HCC. In addition, the use of iron oxide nanoparticles may prove to be successful in tracking and localizing MSCs to the site of the lesion, which may provide a documentation of their therapeutic effect.

Grant Funding Source: Supported by grant from the Deanship of Scientific Research, King Abdulaziz University Jeddah, KSA
Hematin supplementation protects against hyperoxia‐induced airway hyperresponsiveness (870.8)
Authors: Sopi Ramadan , Zaidi Syed , Mladenov Mitko , Temaj Gazmend , Sahiti Floran , Jakupaj Muharrem
Published: June 2021

It was shown that neonatal hyperoxic‐exposure associates with airway hyperresponsiveness and impaired nonadrenergic noncholinergic relaxation. Previously we have demonstrated the ability of endogenous carbon monoxide (CO) to normalize the airway relaxation under hyperoxic conditions. The aim of this study was to investigate the effect of CO on the hyperoxia‐induced airway hyperresponsiveness. Tracheal cylinders were excised from rat pups 12 days old, exposed to hyperoxia or room air for seven days. Electrical field stimulation (EFS) was applied to induce contraction. The preparations were supplemented with CO precursor ‐ hematin in presence or absence of heme oxygenase (HO) inhibitor tin protoporphyrin‐IX (SnPP‐IX; 100 µM), and/or soluble guanylyl cyclase inhibitor 1H‐[1,2,4]oxadiazolo[4,3‐a] quinoxalin‐1‐one (ODQ; 10 µM). In TSM obtained from hyperoxia‐exposed animals the contractile responses were increased significantly as compared to room air‐exposed animals, but supplementation of tissues with hematin reversed the contractile responses in hyperoxic group to almost normal level. Inhibiton of HO by SnPP‐IX diminished this effect of hematin. Similar effect was shown also by ODQ alone or in combination with SnPP‐IX. We conclude that increase of CO production overcomes the effects of hyperoxia on airway contractile responses via cGMP signaling and this may serve as a therapeutic approach for treatment of increased airway reactivity in hyperoxia‐exposed neonates.
Both central and peripheral phrenic‐intercostal circuitry mediate respiratory recovery following cervical spinal cord injury (871.1)
Authors: Awad Basem , Warren Philippa , Alilain Warren
Published: June 2021

The organization of the respiratory network has traditionally been described through descending medullary tracks. We have determined central and peripheral mechanisms auxiliary to the descending supraspinal pathways through which intercostal activity can augment breathing. Following lateral C2 hemisection (LC2Hx), functional recovery of the paralysed ipsilateral diaphragm is largely mediated through the crossed phrenic pathway (CPP). However, the contribution of the intercostals to this effect has not been determined. We show that LC2Hx results in paralysis of the ipsilateral intercostal muscles. However, contralateral phrenicotomy returns activity to both the paralyzed hemidiaphragm and intercostals. These exciting data demonstrate the presence of a crossed intercostal phenomenon acting in synchrony with the CPP. We demonstrate the further interconnectivity of phrenic and intercostal motoneurons across the spinal cord (SC) midline through intercostal‐phrenic ascending pathways. These pathways are necessary for hemidiaphragmatic recovery after LC2Hx. Together, these data are indicative of both descending and ascending bilateral crossed pathways to control respiratory motor activity following cervical trauma. Further to these central mechanisms, we provide evidence of an alternative monosynaptic peripheral pathway between the phrenic motor nucleus and the intercostal muscles, which can maintain intercostal respiratory activity following lower cervical SC transection. In conclusion, significant redundancy acts both centrally and peripherally to the cervical SC to mediate respiratory activity following trauma. These data strongly implicate the necessary role of intercostals in recovery of respiratory motor function following spinal cord injury, as well as alternative therapeutic targets.

Grant Funding Source: Supported by Craig H. Neilsen Foundation and MetroHealth Medical Center
Middle cerebral artery occlusion reduces α7 nicotinic receptor expression whereas α7 nicotinic receptor stimulation suppresses inflammation and promotes microglial Mox phenotype (877.7)
Authors: Hua Sansan , Darsalia Vladimer , Patrone Cesare , Mallard Carina , Johansson Maria
Published: June 2021

Inflammation plays a central role in ischemic stroke. During brain inflammation the resident macrophages of the brain, the microglia cells, are rapidly activated. In the periphery, α7 nicotinic acetylcholine receptors (α7R) present on macrophages can regulate inflammation by suppressing cytokine release. In the current study we investigated the expression α7R in mice after middle cerebral artery occlusion (MCAO). We further examined possible anti‐inflammatory role of α7R stimulation in vitro and microglia polarization after α7R agonist treatment. Real‐time PCR analysis showed a 58% reduction in α7R expression 72 h post MCAO. Stimulation of primary microglial cells with LPS in combination with increasing doses of the selective α7R agonist AR‐R 17779 significantly attenuated TNF‐α release and increased α7R transcript in microglial cells. Gene expression of M1 markers CD86 and iNOS, as well as M2 markers CD206 and Arginase1 was not influenced by LPS and/or α7R agonist treatment, however, Mox markers heme oxygenase (Hmox1) and sulforedoxin‐1 (Srx1) were significantly increased, suggesting a polarization towards the Mox phenotype after α7R stimulation. Thus, our data support the anti‐inflammatory role of α7R in the brain after MCAO and in microglia, suggesting that α7R stimulation could enhance the polarization towards a reparative Mox phenotype.
Left ventricular structure and function in the aging naked mole‐rat, the longest‐lived rodent (879.1)
Authors: Grimes Kelly , Lindsey Merry , Buffenstein Rochelle
Published: June 2021

The extraordinarily long‐lived mouse‐sized naked mole‐rat (NMR) shows an attenuated age‐related decline in diastolic function compared to mice and humans, despite having a maximum lifespan of >31 years. Using echocardiography, we have examined the left ventricles (LVs) of young (2‐5 years), middle‐aged (8‐10 years) and old (16‐22 years) NMRs (n=10/cohort). Young NMRs displayed low LV contractility, as measured by fractional shortening (~28%), which was not significantly changed with age. LV wall thicknesses were significantly greater in young NMRs when compared with physiologically age‐matched mice, and NMR LV wall thickness did not decline with age as has been reported for mice. Dobutamine stress echocardiography (3 μg/g body weight i.p.) elicited similar percent changes in fractional shortening, ejection fraction, and cardiac output in each cohort, indicating that cardiac reserve in the NMR is maintained with age. This is in contrast to reports to age‐related declines in cardiac reserve in both mice and humans. Interestingly, cardiac reserve in NMRs of all ages studied here was considerably greater than cardiac reserve we and others have observed in young adult mice. Further studies will investigate the mechanisms by which the NMR may attenuate declines in cardiovascular composition and function commonly observed during aging in other species.
DNA methylation contributes to chronic hypoxia‐induced downregulation of large‐conductance Ca2+‐activated K+ channels in uterine arteries in pregnancy (889.3)
Authors: Hu Xiang‐Qun , Zhu Ronghui , Zhang Lubo
Published: June 2021

Uterine blood flow increases dramatically during pregnancy in part due to an upregulation of the large‐conductance Ca2+‐activated K+ (BKCa) channel in the uterine artery. However, this hemodynamic change is complicated by chronic hypoxia. We hypothesized that DNA methylation may play a role in chronic hypoxia‐induced downregulation of BKCa channels. Uterine arteries were isolated from nonpregnant (NPUA) and near‐term (~140 days) pregnant (PUA) sheep and incubated under 21% or 10.5% O2 for 48 hours at 37°C. Exposure to chronic hypoxia significantly reduced the BKCa current density in PUA (p < 0.05), but not in NPUA. 5‐aza‐2'‐deoxycytidine (5‐aza), a DNA methylation inhibitor, blocked the hypoxia‐mediated effect in PUA. Accordingly, chronic hypoxia decreased a BKCa activator NS 1619‐induced relaxations of PUA, which was abrogated by 5‐aza. Treatment of NPUA ex vivo under 21% O2 with physiological concentrations of estrogen and progesterone (E2β/P4) resulted in increased BKCa current density; and chronic hypoxia suppressed this upregulation. However, 5‐aza reversed the impact of chronic hypoxia and restored the ability of E2β/P4 in upregulating the BKCa current density. Taken together, our findings suggest that an epigenetic modification of DNA methylation plays an important role in chronic hypoxia‐induced downregulation of BKCa channel function in uterine arteries in pregnancy.

Grant Funding Source: HL089012, HL110125
IGF‐1 acutely increases activity of basolateral ClC‐K2‐like Cl‐ channels in the distal nephron (892.12)
Authors: Zaika Oleg , Mamenko Mykola , Boukelmoune Nabila , Pochynyuk Oleh
Published: June 2021

Insulin growth factor 1 (IGF‐1) is known to promote anti‐natriuresis by stimulating ENaC‐mediated sodium reabsorption in the distal renal tubule. However, it remains unclear whether IGF‐1 also augments Cl‐ transport at this place. Using patch clamp electrophysiology in freshly isolated mouse distal nephrons we detected highly abundant single channel conductance of approximately 14 pS in a cell‐attached configuration. Decreased Cl‐ concentration in the pipette resulted in a rightward shift of I‐V relation, indicating Cl‐ selectivity of the recorded channel. The single channel properties of the recorded channel are comparable with ClC‐K2 that was reported to be functionally expressed predominantly in the intercalated cells of distal nephron. In tandem with apically localized pendrin, ClC‐K2 is thought to contribute to distal nephron Cl‐ absorption. We found that acute application of 100 nM IGF‐1 significantly increases open probability of the ClC‐K2‐like channel in a reversible manner. Pretreatment of distal nephrons with a selective PI3‐kinase inhibitor LY292002 abolished activation of the channel by IGF‐1. We propose that, concomitantly with its stimulatory effects on sodium transport, IGF‐1 also favors distal nephron Cl‐ absorption by increasing activity of basolateral ClC‐K2‐like channels in a PI3‐K‐dependent manner.

Grant Funding Source: DK095029
Low basolateral Na+ pump activity in macula densa cells may be necessary to generate tubuloglomerular feedback responses (892.32)
Authors: Nadal‐Quiros Monica , Nieves‐Gonzalez Aniel , Moore Leon , Marcano Mariano
Published: June 2021

Macula densa (MD) cells sense changes in tubular fluid [Cl–] and this initiates a chain of events within the juxtaglomerular apparatus (i.e., the tubuloglomerular feedback (TGF) response) that regulates GFR. It is known that MD cells have much lower basolateral Na+/K+ATPase activity than neighboring cortical thick ascending limb (cTAL) cells. Also, an increase in luminal [NaCl] leads to the depolarization of the MD cell basolateral membrane, an event thought to be an essential step in the TGF response. To study the role of the Na+ pump in MD cells, we created a mathematical model that includes kinetic and thermodynamic models of the transport proteins and channels in MD cells. We examined the influence of luminal [NaCl] on basolateral membrane potential difference (PD). For comparison, similar computations were made with a model of a cTAL cell. When the luminal NaCl was varied from 20 to 150 mM, the basolateral membrane of the MD cell depolarized from –71 to –47 mV. For the cTAL cell, the basolateral membrane potential remained near –96 mV. These results suggest that the low expression of the basolateral Na+ pump may be appropriate in MD cells because, in these sensory cells, cytosolic [Na+] tracks luminal [Na+]. In a reabsorbing cTAL cell, the high basolateral Na+ pump expression stabilizes cytosolic [Na+] and basolateral PD.

Grant Funding Source: Supported in part by MBRS‐RISE grant 2R25GM061151‐12 and by NIH‐NIGMS grant SC1GM084744.
Posttranscriptional regulation of tristetraprolin and tumor necrosis factor through a two‐compartment model (902.12)
Authors: Aslam Naveed , Mack Wade , Alvi Farah
Published: June 2021

TNF is a pro‐inflammatory cytokine that influences a broad range of immunological responses, including chronic inflammation of the gastrointestinal tract. Prolonged inflammation of gastrointestinal tract is linked with inflammatory bowel disease. TNF biosynthesis is regulated both at transcription and posttranscriptional levels. However, the stimulation‐induced increase in translation rate is much larger. This might indicate the possibility of a posttranscriptional regulatory mechanism. How, during basal conditions, is the free concentration of TNF tightly regulated at low levels in cytosol? The stability and translational efficiency of TNF transcript are regulated by an AU‐rich element (ARE) in the 3′‐UTR of messenger RNA. A transacting protein, TTP, binds to ARE and enhances the mRNA turnover. Here, we examine a proposal that TNF homeostasis is regulated by a two‐compartment interaction loop between TTP and TNF. We propose a computational framework of this regulatory loop by modeling the shuttling of TTP between nuclear and cytosolic compartments. This two‐compartment regulatory loop between TTP and TNF is based on activation, deactivation, and translocation/re‐translocation cycle of TTP protein. The mutual interaction of these two proteins regulates the biosynthesis response of both TTP and TNF during basal and inflammatory conditions.
Increased sensitivity to DSS‐induced colitis in the endoplasmic reticulum stress transducer OASIS deficient mice (902.2)
Authors: Hino Kenta , Saito Atsushi , Asada Rie , Imaizumi Kazunori
Published: June 2021

OASIS is a transmembrane transcription factor which is localized at endoplasmic reticulum (ER), and is cleaved at transmembrane region in response to ER stress. OASIS is expressed in goblet cells, osteoblasts and astrocytes. We have previously showed that the number of goblet cells in the large intestine was decreased in Oasis‐/‐ mice. In the present study, we examined the roles of OASIS in the development of colitis using mouse models induced by dextran sulfate sodium (DSS). Oasis‐/‐ mice induced DSS colitis showed significantly loss of bodyweight and increased mortality compared with those of WT mice. Pathologically, the mucosa of large intestine in Oasis‐/‐ mice exposed to DSS displayed severe erosion and ulceration involving infiltration of inflammatory cells. The expression levels of ER stress markers, Bip and Chop, and cell death markers, cleaved caspase‐3 and cleaved caspase‐12 were increased in the mucosa of large intestine in Oasis‐/‐ mice compared with those of WT mice. These data indicate that the ER stress‐induced apoptosis is accelerated in Oasis‐/‐ mice with DSS colitis. The abnormalities were improved by the treatment of Oasis‐/‐ mice with tauroursodeoxycholic acid, which is one of the chemical chaperones and facilitates protein folding. Taken together, our study suggests that the OASIS plays important roles in protection from ER stress and inflammation in DSS‐induced colitis.
Regulation of phosphate transport by dietary Pi in the small intestine of rats (908.2)
Authors: Candeal Eduardo , Caldas Yupanqui , Levi Moshe , Sorribas Victor
Published: June 2021

Pi homeostasis is tightly controlled by the harmonized action of the intestine, kidney and bone. To date, three Na‐dependent Pi transporters have been found in the small intestine: NaPi2b, Pit‐1 and Pit‐2, but the coordinated modulation of all three by dietary Pi is unclear.

Male Wistar rats were fed ad libitum 0.1% vs. 1.2% Pi diets, for either 1, 5 or 10 days. Brush border membrane vesicles (BBMV) and RNA were obtained from duodenum (D), jejunum (J) and ileum (I).

0.1% Pi diet increased Na‐dependent Pi transport in D and J BBMV at 1, 5 and 10 days, as well as the expression of NaPi2b, but only in J for Pit‐2. Pit‐1 expression was increased only at 10 days in D and J. In the I, Pi transport was very modest and adaptation to 0.1% Pi diet was not observed, however, NaPi2b and Pit‐2 expressions were increased at all three times. The changes in NaPi2b and Pit‐1 protein expressions were confirmed by immunohistochemistry, but the changes in Pit‐2 were not noticeable.

Pi transport was always higher at pH 6.0 than 8.5 but inhibited by phosphonoformic acid. This suggests the existence of a novel Na‐dependent transporter, different from NaPi2b, Pit‐1 and Pit‐2, and regulated within 24 h by dietary Pi.
22q11.21 Deletion associated with sporadic Tetralogy of Fallot in Han Chinese (912.3)
Authors: He Guo‐Wei , Maslen Cheryl L. , Bai Xiao‐Yan , Liu Xiao‐Cheng , Liu Zhi‐Gang , Yang Qin
Published: June 2021

Tetralogy of Fallot (TOF) occurs in one in 3,000 live births and accounts for 10% of all serious congenital heart disease in the U.S. and the incidence is substantially higher in China. We investigated the genetic basis of TOF in Han Chinese by using Affymetrix genome‐wide human SNP array 6.0 in DNA extracted from blood of patients and controls. Copy number was calculated based on probe hybridization signal intensity data relative to the signal of disease‐free normal controls. To determine CNV status on each sample we performed real‐time PCR. TaqManTM probes designed by the manufacturer (Applied Biosystems, Foster City, CA) were used to target each of the specific region. The RNase P was chosen as the reference gene. Deletion of 22q11.21 was seen in 8/60 (13.3%) cases. However, the deletion was seen in different patterns in those patients, shown by different starting and ending loci and involving genes. Parents of TOF probands with a disease‐associated CNV provided with ability to determine if any of the CNVs were inherited or de novo. The CNVs were de novo mutations for all these cases. Thus, 22q11.21 deletion is associated with sporadic tetralogy of Fallot in Han Chinese involving a large number of genes and the related individual genes warrant further detailed investigations.

Supported by NSFC 1170148, 2010CB529500, 2012‐BH110004, 2011BHKZ001, 2012BWKZ 008, and CUHK4774/12, China.
Preservation of the brain in cadavers embalmed according to the Thiel protocol (913.2)
Authors: Filgueira Luis , Jungo Franz , Gagliano Marco , Yotovski Peter
Published: June 2021

Embalming according to the method developed by Walter Thiel has been well accepted world‐wide for practical teaching and training of surgical procedures, as this embalming procedure preserves the softness and elasticity of the tissues. However, this method has the disadvantage of the insufficient preservation of the brain. Walter Thiel has therefore proposed additional intracranial in situ fixation of the brain (W. Thiel, Ann Anat 2002, 184, 267‐9). As the brain is usually taken separately for teaching of neuroanatomy and neurohistology, or for research purpose, we explored the possibility of taking the brain before embalming the body using the Thiel protocol. 4 cadavers were used for this preliminary study. Firstly, the brain was gently excised from the cranial cavity after an extended craniotomy, including the dura mater, the pituitary gland and the medulla oblongata. Thereafter, the internal carotid and vertebral arteries were ligated and the body was perfused using the Thiel protocol. There was no substantial leakage of embalming liquid and the bodies could be used later on for teaching and training procedures. The brains were further processed. They were either freshly processed for research purpose, or fixed in 10% formaldehyde and used either for teaching in the neuroanatomy course or for histology. In conclusion, we recommend that the brain may be taken and processed separately from bodies that are embalmed with the Thiel protocol, especially when human brains are needed for teaching and research purpose.
Rectus capitis posterior minor and its relationship to the spinal dura mater: a histological investigation (913.3)
Authors: Venne Gabriel , Reifel Conrad , Pang Stephen , Ellis Randy
Published: June 2021

The rectus capitis posterior minor (RCPm) is a deep suboccipital muscle connecting the tubercule of the posterior arch of the atlas to the inferior nuchal line of the occipital bone. Although controversial, a number of dissection‐based or imaging‐based reports suggested a direct connection between the RCPm and the dura mater. Detailed histologic investigation is required to establish any link between RCPm and the dura mater.

Left and right RCPms, their bony attachments and adjacent spinal dura mater, were resected from 6 embalmed and 2 unfixed cadavers. Tissue samples were fixed with 4% paraformaldehyde in phosphate buffered saline and decalcified. Both left and right RCPms were subdivided into medial and lateral parts and processed for histological examination. Serial histological sections were prepared in order to include maximum surface area; Masson’s trichrome stain was used to visualize muscle and connective tissue connections.

Histological analysis suggested an indirect link between RCPm and the dura mater. This investigation also found tissue distributions previously unreported, i.e.: periosteum of the occipital bone blends into the spinal dura mater on the medial aspect; tissue between the RCPm and dura mater has a structural organisation similar to a bursa or fat pad and this structure is reinforced by traversing collagen bundles taking origin at the peri‐ and/or endomysium of the RCPm.

This histological investigation clarifies some anatomical features of the suboccipital region and indicates a secondary function of the RCPm, which may include posterior traction of the dura mater during neck hyperextension.

Grant Funding Source: Natural Sciences and Engineering Research Council of Canada (NSERC)