bluuebunny/crossref_metadata_2025 huggingface dataset:
Cardiac Specific Inactivation of ClC‐3 Gene Reveals Cardiac Hypertrophy and Compromised Heart Function
Authors: Xiong Dazhi , Ye Linda , Neveux Iva , Burkin Dean J. , Scowen Paul , Evans Rebecca , Valencik Maria , Duan Dayue , Hume Joseph R.
Published: June 2021

To test the hypothesis that chloride channel ClC‐3 comprises a key component of native volume‐sensitive outwardly rectifying anion channels (VSOAC) in mammalian heart and further assess its physiological role in cardiac function, we generated cardiac‐specific Clcn3 knockout (KO) mice, utilizing heart‐specific α‐myosin heavy chain (α‐MHC) promoter and Cre/Lox technology. Examination of the properties of VSOACs in isolated KO atrial myocytes revealed two groups of cells: in group 1 KO myocytes, VSOAC current densities were very significantly attenuated compared to WT cells, whereas in group 2 KO myocytes, VSOAC current densities were similar to WT cells, but several VSOAC properties were altered, possibly due to compensatory up‐regulation of other unidentified proteins. Electrocardiograms revealed a significantly shortened QRS‐complex in the KO mice, compared to age‐matched wild‐type (WT) mice. Echocardiography analyses revealed dramatically reduced ejection fraction and fractional shortening, and severe signs of myocardial hypertrophy and heart failure in the KO mice. Furthermore, these KO mice had significantly increased heart weights, and heart weight‐body weight ratios, compared to age‐matched WT mice. The results indicate a significant cardioprotective role of ClC‐3 against the development of cardiac hypertrophy and failure. (NIH grants HL‐49254, HL‐063914, and NCRR P20RR1581)
Non Invasive Evaluation of Cardiac Dysfunction in β‐thalassemic Mice
Authors: Stoyanova Ekatherina , Cloutier Guy , Felfly Hady , Trudel Marie
Published: June 2021

Beta‐thalassemia is a genetic hematological disorder characterized by mutations of the hemoglobin gene causing total or partial decrease of the β‐globin chain production. This disease leads to a chronic hemolytic anemia, cardiac complications and blood circulatory problems such as transitory ischemic attacks, thromboembolic events and microcirculatory obstructions. Our objective is to study and dissect the molecular physiologic mechanism of cardiovascular dysfunction in β‐thalassemia. We have generated a murine model of β‐thalassemia major primarily for the investigation of the cardiovascular physiology. Left ventricular morphology and function were assessed by echocardiography in 13.5‐month‐old β‐thalassemic mice and age matched controls. The left ventricular mass index and total peripheral vascular resistance were significantly increased (p < 0.05 and p < 0.01, respectively) in β‐thalassemic mice compared to wildtype controls. Furthermore, fractional shortening and cardiac output index were significantly decreased (p < 0.01) in β‐thalassemic mice compared to age matched controls. Thus, these data in β‐thalassemic mice showed left ventricular remodeling by dilatation and hypertrophy that likely play a key role in cardiac dysfunction.

This work was supported by the NIH (#RO1HL078655), CIHR (#CMI 72323), HSFC (#PG‐05‐0313).
Beneficial effects of folic acid supplementation on left ventricular volume overload
Authors: Rezk Bashir M , Lord Kevin C , Reed Ryan E , Hutchinson Kirk R , Espinoza Carmen , McIlwain Elizabeth , El‐Kak Abd El‐Aziz A , Varner Kurt J , Lucchesi Pamela A
Published: June 2021

Cardiovascular disease is the major cause of worldwide morbidity and mortality. This is the first study to investigate the cardioprotective effects of folic acid (FA) on left ventricular remodeling in response to acute volume overload generated by aorto‐caval fistula (ACF) Adult male Sprague‐Dawley rats were subjected to ACF for 72 hr ± pretreatment with folic acid (30 mg/kg/day) along with sham operated controls. Echocardiography and in vivo pressure volume loops indicated left ventricular (LV) dilation in ACF vs. sham that was associated with oxidative stress (decreased GSH/GSSG ratio), extracellular matrix (ECM) degradation and apoptosis. Interestingly, TUNEL‐positive cells resided only in the microvascular component of the ACF LV. Endothelial cell apoptosis lead to decreased LV capillary density, whereas ECM degradation in response to ACF was associated with mast cell degranulation and increased MT1‐MMP expression compared to sham. Folic acid supplementation remarkably reversed LV dilation, prevented apoptosis, decreased oxidative stress, and increased both VEGF isoform expression and capillary density despite continued volume overload induced by ACF. The prominent cardioprotective effects of FA may be due to its potent antioxidant and proangiogenic activities.
Progression of autonomic changes in heart rate control during development of obesity
Authors: Carroll Joan F , Winterrowd Kim , Heymann Abraham
Published: June 2021

Both acute overeating and chronic obesity increase heart rate (HR), but it is unclear whether autonomic mechanisms of HR control are altered during the progression from acute overeating to chronic obesity. This study used pharmacologic blockade to determine parasympathetic (PSNS) and sympathetic (SNS) influences on HR control during 1 wk of acute overeating vs 12 wks of developing obesity. PSNS and SNS influences on HR control were estimated using blockade by scopolamine (SCOP) and metoprolol (METO), respectively. HR after double blockade represented intrinsic HR. Compared with controls, acutely overfed rabbits had a reduced HR response to SCOP, and a similar HR response to METO. Intrinsic HR tended to be higher (p=0.08). Calculations of autonomic balance indicated reduced PSNS influence (p=0.03). In contrast, obese rabbits had similar increases in HR after SCOP, greater decreases in HR after METO, and increased intrinsic HR. Calculations of autonomic balance indicated both reduced PSNS influence and increased SNS influence, resulting in a significant shift toward SNS control of HR (p=0.04). These data suggest that 1 wk of acute overeating negatively impacted PSNS control of HR, while chronic obesity negatively altered both SNS and PSNS control of HR.
Evaluation of arrhythmia scoring systems and exercise induced cardioprotection following ischemic reperfusion injury
Authors: Quindry John C , Wrieden Jenna , Schrieber Lindsey , Hoyt Emily , Hosick Peter
Published: June 2021

Exercise is protective against ischemia reperfusion (IR) induced arrhythmia. Numerous analytical devices exist arrhythmia quantification. Based preventricular contractions, ventricular tachycardia, and ventricular fibrillation frequency, these scoring systems are intended to provide a more robust indicator electrocardiographic (ECG) outcome than isolated variables. In this investigation 7 ECG evaluation systems were used to assess arrhythmias surgically induced IR (I = 20 min, R = 30 min) in Sprague Dawley rats. ECG data from sedentary and exercised animals (60 min bouts of treadmill exercise 3 days prior to IR) were evaluated under blinded conditions by 3 individuals trained in ECG analysis. One‐way ANOVA was used to compare arrhythmia scores between sham (surgery, no IR), exercised, and sedentary animals. IR produced a significant arrhythmic response in exercised and sedentary rats for all arrhythmia scores. In animals receiving IR, 4 arrhythmia scores resulted in significant differences between exercised and sedentary treatments (P < 0.001) while 3 scores did not. Variations in scoring system data reduction approaches explain why only 4 of 7 identified a protective response in the exercised animals. These data confirm that exercise protects against arrhythmias induced by IR and that care must be taken when selecting an arrhythmia scoring system for ECG evaluation.
Proteomic analysis of changes during the onset of amphibian limb regeneration
Authors: Mescher Anthony Louis , Neff Anton , King Michael
Published: June 2021

Synthesis of specific proteins has been examined during the early phase of limb regeneration, but an overall analysis of protein changes in this period has been lacking. We report the first global proteomic analysis of this system, utilizing LC/MS‐based identification and quantification methods to compare abundant proteins in hindlimbs of the larval anuran Xenopus laevis (stage 53) at the time of amputation (0dPA) and 3 days later when the regeneration blastema is developing (3dPA). We identified and quantified 1517 peptides, of which 1067 were identified with high peptide ID confidence (555 with multiple peptides and 512 with a single peptide). Of these 1067 proteins, 489 showed significant changes in quantity between the two groups. Among those proteins that were elevated in the tissue relative to 0dPA were members of the annexin family (e.g. ANXA1, ANXA2, ANXA5) and the ANXA2‐binding partner S100A10, which have important immunoregulatory roles in other systems and which were also shown to be differentially expressed in stage 53 and 57 3dPA and 5dPA tissues in our previous microarray studies. Quantitative PCR (qPCR) revealed that the differences in annexins and S100A10 protein levels were consistent with the differences in levels of gene expression. Besides elucidating the possible modulation of inflammation during amphibian limb regeneration, the results also provide insight into the dedifferentiation process by revealing up‐regulation of various proteins known to characterize many stem cells. This work was supported by a pilot grant from the Indiana University School of Medicine.
Embryonic exposure to 5‐AZA‐2’‐Deoxycytidine (5‐AZA‐CdR) can affect epididymal sperm numbers of adult CD‐1 mice ‐ Epigenetic Implications
Authors: Cisneros Francisco Javier , Branch Stacy , Hurley Steven , Schreur David , Hoberman Alan
Published: June 2021

Spermatogenesis and spermiogenesis demand adequate expression of specific genes at particular times. DNA methylation controls in part gene expression of mammals from embryogenesis to senescence. Altered epigenetic processes affecting sperm have been reported recently. The adverse effects of 5‐AZA‐CdR in sperm of adult mammals have been substantiated. Yet, its effects on sperm after an in utero insult remain to be elucidated. To investigate if an in utero exposure to 5‐AZA‐CdR – a demethylating agent‐ affects epididymal sperm numbers during adulthood. CD‐1 mice at gestation day 10 were administered 1 mg/kg of 5‐AZA‐CdR via intra peritoneal injection once. Body Weight, testis and epididimides of offspring were collected at post natal day 154. The number of epididymal sperm was determined by Computer Assisted Semen Analysis. Body Weight and epididymal sperm number of exposed mice were statistically lower than controls (P<0.05 and P<0.019 respectively) while relative testis and epididymis weights were not. This suggests that the 5‐AZA‐CdR in utero insult impaired spermatogenesis and/or spermiogenesis reducing the number of sperm present in the epididimides. Further studies are needed to elucidate the effects of altered methylation patterns induced during embryogenesis on sperm viability and/or availability during adulthood and the subsequent impact in the offspring of in utero exposed individuals. NIH‐ES08452 & CRL
The Dynamic Changes Of Caveolin‐1 In Uterus During The Implantation
Authors: Wang Lin , Wang Qianhua
Published: June 2021

OBJECTIVE: The aim of this study was to examine caveolin‐I expression in mouse uterus during the implantation and to explore its effect on implantation. METHODS: To detect the mRNA expression of caveolin‐1 in uterus with semiquantitative reverse transcriptase‐polymerase chain reaction technique. At the same time, to detect the expression of caveolin‐1 in uterus with immunohistochemistry technique. RESULTS: The mRNA expression of caveolin‐1 was significantly higher in endometrial decidual tissue on day 9.5 of pregnancy than that of day 3.5 to 7.5 of pregnancy and non‐pregnancy endometrial decidual tissue. The differences were significant (P<0.05). A strong level of caveolin‐1 expression was observed in the decidualized cells and vascular endothelial cells at mesometrium side from days 4.5 to 9.5 of pregnancy. The positive expression of caveolin‐1 can be observed in cytotrophoblast cell but not in syncytotrophoblast cell. CONCLUSION: In conclusion, these results suggest that caveolin‐1 expression may be closely related to embryo implantation and decidualization by regulating vascular permeability and angiogenesis.
Mechanistic study on binding of human Rad54 to nucleosome
Authors: Wang Yuzhen , Tsai Yu‐Cheng , Urena‐Rivera Esteban , Chen Junghuei
Published: June 2021

How to make DNA accessible to hRad51 or hRad51‐ssDNA filament during HR process is still a puzzle, although several studies showed that Rad54 exhibit chromatin remodeling activity. Here we explore interaction of Rad54 with nucleosomes. N‐His‐tagged human Rad54 and N‐His plus C‐Flag‐tagged human Rad54B were purified through baculovirus system. Various biochemical assays were performed to characterize hRad54 and hRad54B. We found that hRad54's ATPase activitity is more efficient than hRad54B's, and significantly decreased in the presence of nucleosome, no such change was observed with Rad54B. We also found that hRad54, not hRad54B, exhibits strong binding activity to nucleosomes, preferentially binding to high order aggregates of nucleosomes in low concentration. No such strong binding shift on naked DNA was observed with either hRad54 or hRad54B in the same condition. We are currently preparing N‐terminal truncated Rad54 which lack the primary binding site of Rad51, and C‐terminal truncated Rad54 with deletion of positively charged surface patch, and several single mutants selected in conserved DNA binding site and bi‐lobe ATPase core to further characterize the binding mechanism of hRad54 with nucleosomes. In addition, the discrepancy in ATPase activity and nucleosome binding between hRad54 and Rad54B is being confirmed with untagged proteins.
Investigating Msh2‐Msh3 Function in DNA Mismatch Repair
Authors: Biro Francis Noah , Hingorani Manju
Published: June 2021

DNA mismatch repair (MMR) is an evolutionarily conserved process that corrects errors such as base‐base mismatches and insertion/deletion loops (IDLs) in DNA. MutS proteins (eukaryotic MutS homologues) bind errors in DNA and initiate the repair process. In humans and other eukaryotes, Msh2‐Msh6 complex recognizes base‐base mismatches and small IDLs, and Msh2‐Msh3 complex putatively recognizes both small and large IDLs. Available evidence suggests that Msh2‐Msh3 loop‐binding activity is involved in aberrant triplet‐repeat expansion, which is implicated in such neurodegenerative disorders as Huntington's disease. In order to better understand the function of this protein, we have overexpressed and purified S. cerevisiae Msh2‐Msh3 from E. coli cells. We are currently measuring the kinetic and thermodynamic parameters that govern Msh2‐Msh3 DNA binding and ATPase activity and define its mechanism of action in MMR.
Pat1 contains distinct functional domains that promote translational repression, P‐body assembly and activation of decapping
Authors: Pilkington Guy Robert , Nissan Tracy , Parker Roy
Published: June 2021

The control of mRNA degradation and translation are important aspects of gene regulation. Recent results suggest that translation repression and mRNA decapping can be intertwined and involve the formation of a quiescent mRNP, which can accumulate in cytoplasmic foci referred to as P‐bodies. The Pat1 protein is a key component of this complex and an important activator of decapping yet little is known about its function. In this work we analyze Pat1 in Saccharomyces cerevisae function by deletion and functional analyses. Our results identify two primary functional domains in Pat1, one promotes translation repression and P‐body assembly, and a second domain promoting mRNA decapping after assembly of the mRNA into a P‐body mRNP. In addition, we provide evidence that Pat1 binds RNA and has numerous domain specific interactions with mRNA decapping factors. These results indicate that Pat1 is an RNA binding protein and a multi‐domain protein that functions at multiple stages in the process of translation repression and mRNA decapping. Current experiments are directed at understanding the mechanism by which Pat1 affects translation repression and possible roles for nuclear‐cytoplasmic shuttling of Pat1.

NIH grant (R37 GM45443) and funds from the Howard Hughes Medical Institute supported this work.
Function and Regulation of Plasma Glutathione Peroxidase
Authors: Ottaviano Filomena G. , Handy Diane E. , Loscalzo Joseph
Published: June 2021

GPx‐3 is a selenocysteine‐containing protein that catalyzes the reduction of hydrogen peroxide and lipid hydroperoxides using glutathione as a reducing agent (GSH). Our goals are to overexpress GPx‐3, determine its activity, and study its regulation. The complex expression of selenoproteins requires specific translational cofactors (SBP2, SelD, and tRNASec), adequate selenium, and the selenocysteine insertion sequence (SECIS), which facilitate read‐through of the UGA codon for selenocysteine (Sec) and incorporation in the growing polypeptide. Stable Cos 7 cell lines of GPx‐3 cDNA/SECIS, His/V5 tagged wild type, SECIS containing mutated 3'UTR fragments, and a Sec → Cys mutant were generated and grown in medium with adequate sodium selenite. Purification was accomplished by immobilized metal ion affinity chromatography with nickel Sepharose that binds GPx‐3 His tagged protein, and followed by Western blot using an anti‐V5 antibody, and by His‐tag ELISA. The mRNA stability of both the wild type and Cys mutant were examined by Real‐Time PCR. Translation of the wild type GPx‐3 was much less efficient than that of the Cys mutant. Mass spectrometry confirmed GPx‐3 identity. The 3' UTR of GPx‐3 is important in regulating its mRNA stability and protein expression.

Support: HL61795, HV28718, and HL81857
Teaching the microscopic anatomy of the respiratory system to medical students
Authors: Benjamin Michael
Published: June 2021

The talk will cover the approach of the author to (a) delivering a 1 hour lecture on the lower respiratory tract to a large (300+) class of UK medical students in the early part of their course (b) providing suitable support material that can be accessed by the students on a University intranet for self‐directed learning (c) designing a practical class that reinforces points made in the lecture and encourages student‐centred learning. The student lecture is illustrated throughout with points of functional and clinical significance and several examples will be given in the talk. Extensive use is made of simple line drawings of tracheal and lung histology, made by the author using a vector drawing package and a drawing tablet. Although some histological images are shown in the lecture as well, these are largely a feature of the practical class. Students can access all the line drawings used in the lecture on the intranet afterwards (with no copyright issues for the University). With ever increasing student numbers that place a strain on departmental microscope and slide resources (and staff time), our current preference is for developing a database of digital histology images for use in practicals. Students are required to make simple interpretative drawings of histology images, answer questions and screen‐capture and label images.
Clinical Diffuse Lung Injury and Remodeling
Authors: Jobe Alan H.
Published: June 2021

Lung structural abnormalities result primarily from two mechanisms: developmental abnormalities and lung injury with repair. The timing of distal airway and alveolar/vascular development in the human will be reviewed. Developmental disruptions such as renal dysplasia and congenital diaphragmatic hernia that result in pulmonary hypoplasia are relatively common causes of severe lung structural abnormalities. Preterm delivery can interfere with distal parenchymal development and alveolarization, which normally begins in the human at 32‐36 wks gestation. Clinical interventions such as mechanical ventilation and oxygen exposure of the preterm lung interfere with lung development and initiate injury/repair that results in a decreased alveolar septation and decreased microvascular development syndrome called bronchopulmonary dysplasia. Although alveolar septation has been thought to be complete by early childhood, new results suggest that new alveoli can form in the adult lung. As proof of principle, the adult rodent lung can develop emphysema with starvation, and the emphysema disappears with brief refeeding. Diffuse injuries to the mature lung caused by Acute Respiratory Distress Syndrome can resolve remarkably if fibrosis does not occur. The challenges are to learn how to promote alveolar/vascular regrowth and suppress fibrosis following lung injury.
Sustainable TRPM4 Channel Activity in Freshly‐Isolated Cerebral Artery Smooth Muscle Cells
Authors: Gonzales Albert Louis , Amberg Gregory C. , Crnich Rachael , Earley Scott
Published: June 2021

Localized control of blood flow and arterial constriction in response to changes in intraluminal pressure are dependent on the intrinsic regulation of smooth muscle cell membrane potential. The melastatin transient receptor potential (TRP) channel TRPM4 is highly selective for monovalent cations, is activated by elevated levels of intracellular Ca2+, and is a critical regulator of smooth muscle membrane depolarization, which serves to open voltage dependent Ca2+channels (VDCC), and resulting in myocyte contraction. Interestingly, Ca2+ activates and inactivates TRPM4, causing the channel to rapidly inactivate under conventional whole‐cell patch clamp conditions. When the slow Ca2+ buffer, ethylene glycol‐bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid (EGTA), was included in the pipette solution, we identified a transient tetraethylammonium (TEA)‐insensitive inward Na+ current in freshly isolated cerebral arterial myocytes. siRNA‐mediated silencing of TRPM4 and the recently described TRPM4 inhibitor, 9‐phenanthrol, both diminish the open probability (NPo) compared to controls, strongly suggesting the molecular identity of the channel to be TRPM4. This study demonstrates a practical method to record sustained TRPM4 activity and allows for further investigation into mechanisms of channel regulation. AHA0535226N; F31HL094145‐01.
Synthesis of phosphorescence based Oxygen sensors for application in hypoxic environments using multiphoton microscopy
Authors: Doan Julie Angela , Chen Jin , Farnum Cornelia , Williams Rebecca , Schmehl Russell H.
Published: June 2021

The recent demonstration that the transcription factor Sox9, known to be required for chondrogenic differentiation, has significantly different levels of expression depending upon O2 levels, and that Sox9 expression is directly regulated by HIF1α is evidence at the molecular level of the fundamental importance of the degree of hypoxia as a regulator of skeletogenesis. The lack of an ability of make direct O2 measurements in the living animal in experimental systems during either pre‐natal skeletal development in embryos or during post‐natal skeletal development has meant that the potential significance of O2 microgradients in the ECM of differentiating cartilage and the surrounding vasculature has remained largely unexplored in vivo. Our approach is to develop phosphorescent complexes of Ru(II) that can be photoexcited by two photon absorption (λex=800 nm) and exhibit O2 sensitive emission in the red (600‐800 nm). Further, the complexes exhibit varying degrees of oxygen sensitivity for application in environments of differing degrees of hypoxia. The synthesis, photophysical characterization and preliminary in vivo studies of the complexes will be reported.
Hypoxic HK2 Gene Regulation Involves Multiple Distinct Cis‐Acting Elements That Differ In Their Hypoxia‐Inducible Factor‐α (HIFα) Isoform Selectivity
Authors: Robey R. Brooks , Zhong Lihui , Keyes Lauren , Montes Jessica , Zhang Hongmei , Granner Daryl , Printz Richard
Published: June 2021

As the only effective energy substrate in the absence of O2, glucose (Glc) is central to all cellular strategies for hypoxic adaptation. Obligatory reliance on Glc also explains associated induction of glycolytic enzymes such as hexokinases which catalyze the first committed step of Glc metabolism. HIF1α contributes to HK2 expression in cancer cells, but the specific cis‐acting sequences are undefined and corresponding effects in nontransformed cells are unexplored. We therefore used chimeric reporter genes to delineate hypoxia‐responsive cis‐acting HK2 promoter regions in nontransformed NRK‐52E cells.

Major hypoxic control of HK2 expression mapped to a phylogenetically conserved tandem pair of canonical HIF‐binding motifs just 5' to the 157 bp basal promoter region. Targeted mutagenesis of either motif abrogated hypoxia‐responsiveness, as did their spatial separation by a half (+5 bp) ‐ but not a full (+10 bp) ‐ helical turn. Similar results with hypoxia‐mimetics (e.g. CoCl2) and stabilized mutants of HIF1α ‐ but not HIF2α ‐ suggest HIF1α selectivity. Independent hypoxic enhancer activity responsive to both HIF1α and HIF2α ‐ but not other known HK2 transcriptional regulators ‐ was also observed within 90 bp of the transcription start site. These results suggest complex hypoxic gene regulation involving both combinatorial interactions and cis‐acting elements with differing HIFα isoform specificities.
Nox‐4 siRNA Causes Attenuation of Hypoxic Pulmonary Vasoconstriction in Bovine Pulmonary Arteries
Authors: Ahmad Mansoor , Kelly Melissa R , Kandhi Sharath , Zhao Xiangmin , Wolin Michael S
Published: June 2021

Hypoxic pulmonary vasoconstriction (HPV) in endothelium removed bovine pulmonary arteries (BPA) is potentially mediated through the removal of a superoxide‐derived hydrogen peroxide mediated basal relaxation. To study the source of the superoxide, endothelium removed BPA rings were cultured for 48 hrs with the first 24 in the presence of either Nox‐4 or Nox‐2 siRNA. Nox‐4 siRNA caused a significant decrease in Nox‐4 expression without any effect on Nox‐2 levels. Superoxide levels and HPV were significantly reduced in BPA rings treated with Nox‐4 siRNA without affecting force development to KCl. Superoxide levels were significantly reduced with Nox‐2 siRNA treatment however, HPV and KCl force were not affected. Endothelium removed BPA rings treated with apocynin and gp91‐dstat show a decrease in superoxide levels but did not affect either KCl force or HPV. Thus Nox‐4 and not Nox‐2 may be the source for the potential superoxide derived peroxide mechanism maintaining basal relaxation during normoxia which when removed leads to HPV.

Support by NIH grants HL31069, HL43023 and HL66331.
Identifying Diagnostic Markers and Studying Etiologic Factors of Inflammatory Bowel Diseases Using a High‐Throughput Proteomic Approach
Authors: LI XIAOXIAO , LeBlanc James , Goodglick Lee , Elashoff David , Presley Laura , Borneman James , Braun Jonathan
Published: June 2021

The objectives of our study are to identify diagnostic markers for Inflammatory Bowel Diseases (IBDs), define the features of the proteome at different colonic locations and under different physiologic conditions, and investigate the host and microbial interactome at the mucosal interface. We collected bowel‐rinse samples from IBD patients and normal individuals, and analyzed each sample using high‐throughput surface enhanced laser desorption/ionization‐time of flight (SELDI‐TOF) mass spectrometry (MS). SELDI data preprocessing was done in CiphergenExpress®. The resulting spectra were further analyzed in R. Peaks were compared between groups to search for biomarker candidates, whereas the Permutation analysis was used to evaluate the featured differences between groups of samples. Hierarchical clustering heat maps were generated in Cluster3.0 and visualized in TreeView. We also used oligonucleotide fingerprinting of ribosomal RNA genes (OFRG) to study the microbes in the bowel‐rinse samples. We have thus far identified several potential IBD biomarkers which are confirmed by western blotting. The heat‐map showed discriminative protein/peptide patterns in different locations of the colon, and between IBD patients and normal individuals. Preliminary OFRG study suggested that certain types of microorganism are highly associated with certain secreted protein/peptide in the colon.
Requirements for the binding of C‐reactive protein to oxidized low‐density lipoprotein
Authors: Agrawal Alok , Singh Sanjay K. , Thompson James A. , Hammond David J. , Rusinol Antonio E.
Published: June 2021

We are investigating the interactions between C‐reactive protein (CRP) and various forms of low‐density lipoprotein (LDL). Previously, we reported that phosphoethanolamine (PEt)‐complexed CRP, but not free CRP, bound to native LDL and that PEt‐complexed CRP was more potent than free CRP in binding to enzymatically‐modified LDL (E‐LDL). In this study, we explored the binding of CRP to oxidized LDL (oxLDL). In an ELISA‐based solid‐phase binding assay, freshly‐purified native CRP did not bind to immobilized oxLDL. Interestingly, in the presence of PEt, freshly‐purified native CRP bound to immobilized oxLDL efficiently in a dose‐dependent manner. In contrast, when an old preparation of frozen CRP was thawed and used in the assay, CRP bound to oxLDL even in the absence of PEt and the binding was dramatically enhanced in the presence of PEt. The mechanism of action of PEt in the binding of CRP to various forms of LDL is not known, but it is reasonable to assume that PEt modifies CRP in a way similar to storage‐induced modification of CRP. Defining the requirements for the binding of CRP to various forms of LDL is important because, by binding to LDL, CRP can prevent formation of LDL‐loaded macrophage foam cells. As shown previously by using CRP‐E‐LDL complexes, CRP‐bound E‐LDL does not cause formation of foam cells which is one of the processes that contribute to the development of atherosclerosis. NIH R01HL071233
Reactive oxygen and nitrogen species in leukocyte adhesion and transmigration: a computational model
Authors: Lakshmanan Nisha , Kavdia Mahendra
Published: June 2021

In hyperglycemia, there is a significant increase in the levels of tyrosine nitration causing an increase in circulating adhesion molecules. This leads to a greater number of leukocytes that roll and adhere on the vascular endothelium resulting in endothelial cell dysfunction. Some of the adhered leukocytes also cross the endothelial junction barrier and migrate from the lumen to the surrounding spaces. Inducible nitric oxide synthase in these leukocytes produces nitric oxide and superoxide, thereby producing more of peroxynitrite and nitrotyrosine. To better understand this vicious cycle, we formulated a mathematical model of a tissue containing a blood vessel and simulated varying degrees of leukocyte adhesion and transmigration. The model predicts an increase in the peak levels of nitric oxide, superoxide and peroxynitrite at and around the site of adhesion; from 83, 2 and 3.5 nM respectively, in the case of no adhesion to 104, 13.5 and 35 nM when leukocytes adhere. However, the extent of transport is a few microns for superoxide to 10's of microns for peroxynitrite. Results indicate that peroxynitrite can cause tissue injury over a wide area across the adhesion site and trigger further leukocyte adhesion. The model helps elucidate some important aspects of complex leukocyte‐endothelium interactions such as signaling, injury and adhesive response.

Supported by: AHA 0530050N and NIH R01 HL084337 & R15 HL087287.
Paraplegia increases cardiac nerve growth factor content, sympathetic innervation density and the susceptibility to ventricular arrhythmias
Authors: Lujan Heidi L. , Chen Ying , Peduzzi‐Nelson Jean , DiCarlo Stephen E.
Published: June 2021

Many complications associated with paraplegia (e.g. spasticity, autonomic dysreflexia, neurogenic bladder) are mediated, in part, by plasticity within the autonomic nervous system. An under investigated complication associated with paraplegia is an increased susceptibility to ventricular arrhythmias. Therefore, we tested the hypothesis that paraplegia‐induced plasticity within the autonomic nervous system is associated with an increased susceptibility to ventricular arrhythmias. Cardiac nerve growth factor content (ELISA), cardiac sympathetic nerve density (immunopositive for tyrosine hydroxylase) and the susceptibility to arrhythmias (coronary artery occlusion) were determined in intact and paraplegic (4 weeks post transection) rats. Paraplegia, compared to intact, increased cardiac nerve growth factor content (2667 ± 326 pg/ml vs 333 ± 51 pg/ml), cardiac sympathetic innervation density (78 μm2/mm2 vs 23 μm2/mm2), and decreased the ventricular arrhythmia threshold (3.6 ± 0.2 min vs 4.9 ± 0.2 min). Thus, neuroplasticity of autonomic behavior increases the susceptibility to ventricular arrhythmias in paraplegic rats. Supported by HL088615
Regulation of acid signaling by PAR2 in rat vagal pulmonary sensory nerves
Authors: Gu Qihai , Lee Lu‐Yuan
Published: June 2021

Protease‐activated receptor‐2 (PAR2) is involved in airway inflammation and airway hyperresponsiveness, the prominent features of asthma. Airway acidification has been consistently observed in airway inflammatory conditions, and is known to cause cardiorespiratory symptoms that are at least in part mediated through the activation of bronchopulmonary C‐fibers and the subsequent reflexes. This study was carried out to investigate the effect of PAR2 activation on the acid signaling in vagal pulmonary C‐fibers that may occur during airway inflammation. Our in‐vivo study showed that, in anesthetized and spontaneously breathing rats, intratracheal instillation of trypsin (1 mg/ml, 0.1 ml), an activator of PAR2, significantly enhanced the chemoreflex responses to right‐atrial injection of lactic acid. In isolated pulmonary C‐neurons, pre‐incubation of SLIGRL‐NH2 (100 μM, 2 min), a specific PAR2 activating peptide, markedly potentiated the whole‐cell inward currents evoked by extracellular acidification. In addition, a similar sensitizing effect was observed when the neurons were pretreated with 2‐furoyl‐LIGRL‐NH2 (100 μM, 2 min), a newly reported potent and selective PAR2 agonist. In conclusion, activation of PAR2 modulates the acid signaling in pulmonary C‐fiber sensory nerves, and the interaction may contribute to the pathogenesis of asthma and other airway inflammatory diseases. (Supported by grants from NIH, AHA and Parker B. Francis Fellowship in Pulmonary Research)
Effects of chewing gum on specific macronutrient and total caloric intake in an afternoon snack
Authors: Geiselman Paula Jeanne , Martin Corby , Coulon Sandra , Ryan Donna , Apperson Megan
Published: June 2021

Hetherington and Boyland (2007) have reported that gum chewing in the afternoon after lunch produced a significant reduction in subsequent snack intake, compared to the control condition. The objective of the present study was to verify this observation and to assess specific macronutrient intake and total caloric intake after having chewed gum as compared with intake in the control, no‐gum condition. After consuming a standard lunch, subjects chewed gum for 15 minutes hourly for 3 hours and then were tested in a modified version of our Macronutrient Self‐Selection Paradigm (MSSP). The MSSP consisted of 12 snack foods in which macronutrient content was varied significantly and systematically in a 2 (Fat factor: High Fat and Low Fat) X 2 (Carbohydrate Factor: High Sugar and High Complex Carbohydrate) design. Results showed that, in the gum condition, subjects specifically decreased their caloric intake of high‐sugar, snack foods in comparison with their intake in the no‐gum condition. Intake of the other foods presented in the MSSP did not differ between the two conditions. These results suggest that chewing gum may be effective in the control of intake of sweet snacks and thereby would be useful in weight management. Funding provided by a research grant from the Wrigley Science Institute.
Effect of viscosity, protein and dietary fibre in beverages on perceived satiety
Authors: Lyly Marika , Ohls Nora , Salmenkallio‐Marttila Marjatta , Lähteenmäki Liisa , Liukkonen Kirsi‐Helena , Poutanen Kaisa
Published: June 2021

The intake of dietary fibre and protein has been linked with increased satiety and weight management. The aim of this study was to investigate the effect of viscosity, dietary fibre and protein on perceived satiety in beverages. Two different levels of carboxymethyl celluloce (CMC), (0 and 5 g); and two levels of whey protein (0 and 20 g) at energy level 700 kJ were used. The dietary fibre was either high‐viscosity CMC or low‐viscosity CMC. Satiety‐related perceptions of 29 healthy volunteers were measured with line scales for 3 hours after ingesting a sample. The area under curve (AUC; cm*min) was calculated.

Dietary fibre and whey protein increased perceived satiety significantly and independently. The sample containing both dietary fibre and whey protein had the greatest effect on perceived satiety. Higher viscosity showed a tendency to increase satiety compared to the samples with lower viscosity (p=0.091). Dietary fibre and protein in beverages have thus potential to affect weight management.

TEKES (Finnish Funding Agency for Technology and Innovation) partly funded this research.
Responses of thoracic spinal interneurons to vestibular stimulation in decerebrate cats
Authors: Miller Derek M , Mehta Amar S , Mehta Ajeet S , Reighard Derek A , Yates Bill J
Published: June 2021

Stimulation of vestibular receptors produces changes in respiratory muscle activity as well as alterations in sympathetic outflow. Unlike vestibular reflexes acting on the limbs, vestibulo‐respiratory and ‐sympathetic responses are elicited by otolith organ inputs, particularly those generated by body rotations in the pitch plane. Recent studies have shown that thoracic interneurons make connections with respiratory motoneurons and sympathetic preganglionic neurons, but the responses of these cells to vestibular stimulation have not been explored. In the present study, recordings were made from thoracic interneurons in decerebrate, paralyzed cats. 60% of thoracic interneurons were activated by electrical stimulation of vestibular afferents at short latency. The majority of these cells exhibited response dynamics similar to those of otolith organs. 75% of the units were activated by tilts in the roll plane or the planes of the vertical semicircular canals; few cells responded best to pitch rotations. These data show that vestibular signals to thoracic interneurons are distinct from those transmitted to the cervical and lumbar spinal cord; however the spatial properties of the responses of the cells to body rotation differ from that of respiratory and sympathetic outflow. Presumably, the latter responses are synthesized through the integration of signals.
Tumor necrosis factor alpha (TNFα) potentiates pulmonary chemoreflex responses in anesthetized rats
Authors: Lin Ruei‐Lung , Lee Lu‐Yuan
Published: June 2021

TNFa, a pro‐inflammatory cytokine, is involved in the pathogenesis of asthma and also known for its sensitizing effect on somatic nociceptive afferents. This study was carried out in anesthetized rats to test whether TNFa induces pulmonary C‐fiber hypersensitivity. Before intratracheal (i.t.) instillation of TNFa (~6.8 μg/kg; 1 μg = ~105 units), right atrial injection of a low dose of capsaicin (0.5 μg/kg) elicited a very mild respiratory and cardiovascular depression. Thirty min after the TNFa treatment, the same dose of capsaicin triggered a longer apnea; the apneic ratio (the ratio between apneic duration and average expiratory duration) increased from 180 ± 43% before to 633 ± 67% after TNFa (n = 6; P < 0.005). This sensitizing effect of TNFa gradually declined, but lasted for > 120 min. In contrast, vehicle administered in the same manner did not generate any increase in the responses to capsaicin at the same time point. Moreover, in single‐fiber recording experiments, inhalation of aerosolized TNFa (10 µg/ml; ~0.2 ml) caused a significant increase in the responses to injection of the the same dose of capsaicin. These results suggest that TNFα induces an acute, intense and reversible potentiating effect on the pulmonary chemoreflex to capsaicin, which is probably caused by an increase in the sensitivity of pulmonary C‐fibers. (Supported in part by NIH grant HL58686)
Phrenicotomy alters expression of long term facilitation (LTF) in anesthetized rats
Authors: Sandhu Milapjit Singh , Reier Paul J , Fregosi Ralph F , Fuller David D
Published: June 2021

Intermittent hypoxia (IH) can induce a persistent increase in phrenic motor output known as LTF. The cut, central end of the phrenic nerve is typically used to record phrenic motor output, but the impact of phrenicotomy (PhrX) on LTF has not been described. Both the removal of afferent inputs to phrenic motoneurons and alterations in motoneuron excitability secondary to axotomy could alter the motor output recorded from cut phrenic nerves. Accordingly, we tested the hypothesis that PhrX influences LTF following IH. Bilateral phrenic neurograms were recorded in anesthetized, ventilated and vagotomized male Sprague‐Dawley rats before (i.e. baseline), during and after 3, 5‐min bouts of isocapnic hypoxia (PaO2 = 30‐50mmHg) in phrenic intact (n=5) and PhrX rats (n=5). At 60 minutes post‐hypoxia, phrenic burst amplitude was significantly greater than baseline (i.e. LTF was present, p<0.05) in PhrX recordings (151±22% baseline) but not in phrenic intact recordings (109±10% baseline). These preliminary studies suggest that removal of phrenic afferents and/or axotomy‐induced changes in phrenic motoneuron excitability may play a role in LTF in anesthetized rats. Funding: NIH 1R01HD052682‐01A1 (DDF) and NIH RO1 NS054025 (PJR).
In vivo functional interactions and computational models of a distributed ponto‐medullary cough network
Authors: Morris Kendall Francis , Davenport Paul W. , Baekey David M. , Nuding Sarah C. , Lindsey Bruce G. , O'Connor Russell , Segers Lauren S. , Shannon Roger , Bolser Donald C.
Published: June 2021

The respiratory network of the medulla reconfigures to produce cough. The aims of this study were to a) identify putative modulatory neurons in behavioral control assemblies (BCAs) specific to cough, and b) model the cough network. Neurons in the ventrolateral respiratory column (VRC), pons, raphe, and a novel caudal medial medullary population (CMM) were recorded with electrode arrays in ten decerebrated paralyzed cats. Neurons in all 4 regions underwent alterations in discharge during coughing. Most pontine, raphe and CMM neurons had little respiratory modulation during fictive breathing but were recruited or strongly modulated during fictive coughing. Cross‐correlation analysis suggested shared excitation between raphe/CMM and VRC neurons, excitation of raphe/CMM neurons by VRC cells, and inhibitory and excitatory effects of raphe/CMM neurons on VRC cells. Cough triggered in network simulations of hyperventilation apnea was followed by transient respiratory cycling; a prediction subsequently observed in vivo. These results also support an important role for non‐respiratory modulated ponto‐medullary neurons in cough BCAs. Support:HL89104, HL89071, NS19814
Seasonally altered GABAA receptors in medullary cardiorespiratory nuclei make neurons unresponsive to high doses of pentobarbital in hibernating, but not summer active, ground squirrels
Authors: Hengen Keith B. , Johnson Stephen M. , Carey Hannah V. , Behan Mary
Published: June 2021

The 13‐lined ground squirrel (Spermophilus tridecemlineatus), a hibernating species, is a natural model of extreme physiology. In torpor, body temperature drops to 0‐4 °C and the cortex is electrically silent, but the brainstem continues to regulate cardiorespiratory function. We hypothesize that this differential regulation of neuronal function is due to altered GABAA receptor function. To test this hypothesis, neuronal firing rates were recorded from medullary and cortical slices from aroused hibernators and summer active squirrels using multichannel recording techniques. During the hibernation season, medullary neuron firing rates were unchanged by pentobarbital (300 μM), whereas cortical neurons stopped firing action potentials. We confirmed that pentobarbital acts on cortical GABAA receptors. Further, we show that functional GABAA receptors are present in the medulla (NTS and VRG) during hibernation and that receptors' sensitivity to GABA does not change. Western blot analyses show that GABAA receptors undergo a seasonal modification in subunit composition. During hibernation, α5 subunit expression is increased in cortex and δis increased in medulla, with posttranslational modification of the δsubunit. These data provide insights into natural mechanisms whereby brainstem function can be sustained in extreme conditions and clinically relevant pharmacological challenges.
Serotonin (5‐HT) Innervation of the Nucleus Tractus Solitarius (NTS) Is Necessary for Sympathetic Recovery following Hypotensive Hemorrhage.
Authors: Kung Ling‐Hsuan , Scrogin Karie
Published: June 2021

We tested if 5‐HT projections to the NTS facilitate sympathetic and respiratory recovery from hypotensive hemorrhage (HH). Rats were subjected to selective (n=9) or sham (n=8) lesion of 5‐HT nerve terminals by injection of 5,7‐dihyroxytryptamine or ascorbic acid (0.01%) into cardio‐respiratory afferent terminal fields of the NTS. After 1 wk, blood pressure (BP), heart rate (HR), renal sympathetic‐ and diaphragmatic EMG activity were recorded during blood withdrawal (21 ml/kg/10 min) in unanesthetized rats. Lesion reduced the initial sympathoexcitation (‐12.3 ± 23.4 vs. +106.6 ± 38.3 % baseline, P<0.05), but not the subsequent sympathoinhibition to blood loss. Sympathetic and ventilatory recovery 35 min after HH were reduced (+6.2 ± 13.2 vs. +127.9 ± 48.7 %baseline, P<0.05 and ‐32.4 ± 4.6 vs. +45.7 ± 18.9 %baseline intergrated dEMG, P<0.05). BP and HR were not affected. Lesion reduced 5‐HT immunoreactivity (IR) in the NTS by 93% (P<0.01), but did not affect tryptophan hydroxylase IR in raphe nuclei or tyrosine hydroxylase IR in adrenergic nuclei. 5‐HT innervation of the NTS is necessary for normal sympathoexcitatory and ventilatory responses to blood loss. Supported by HLBI 72354 and AHA 0815529G.
Gender and age dependent differences in the renal NMDA receptor
Authors: Mandler Laura , Munger Karen A
Published: June 2021

NMDA is a "glutamate" receptor found throughout the brain. We have previously reported functional n‐methyl‐d‐aspartate (NMDA) receptors in rat kidney (J. Am Soc Neph 13:1381, 2002). These calcium channel receptors are independent of renal innervation. NMDA is required for glycine‐induced increase in glomerular filtration rate and renal plasma flow, and also for the increases in oxygen consumption and proximal tubule sodium transport. In this study, we investigated age and gender differences of the various NMDA receptor subtypes in the rat kidney. We measured subtypes, NR1, NR2A‐D, and NR3A‐B in rat kidneys by Western blot (when antibody available) and by RT‐PCR. In addition we looked at NMDA receptor expression in fixed kidney using immunohistochemistry. Results: We found varied amounts of receptor subtypes expression by RT‐PCR and Western Blot. Specifically, we found the NR2A subunit to be significantly lower in female rats compared to males, and gender and age differences in histological expression of NMDA subtypes. This is in agreement with studies finding gender differences in location of the NR2A subunit in the rat brain, and changes in NMDA subtype expression during brain development. These studies reiterate the importance of the renal NMDA receptors and point to another gender‐dependent difference in renal function. Funded by NIH NIDDK RO‐1 DK 62831.