bluuebunny/crossref_metadata_2025 huggingface dataset:
A comparison of the cardiovascular responses to anemia in digesting Burmese pythons and American alligators
Authors: Slay Christopher E. , Enok Sanne , Wang Tobias , Hicks James W.
Published: June 2021

Burmese pythons (Python molurus) and American alligators (Alligator mississippiensis) utilize intermittent feeding strategies, with prolonged fasts punctuated by bouts of voracious feeding. Both species exhibit large increases in postprandial oxygen consumption (V̇O2). Presumably in response to increased O2 demand, P. molurus has been shown to exhibit postprandial cardiac hypertrophy (PCH) of up to 40%. By contrast, alligators exhibit no significant PCH. The “triggers” and universality (both within and between species) of this response therefore remain unclear. Notably, maximal V̇O2 in digesting Burmese pythons may exceed V̇O2 during maximal activity, whereas the opposite is true in alligators. We hypothesize that an O2 supply‐demand mismatch triggers PCH, which was tested in both species by halving hematocrit values (inducing anemia) and feeding animals large meals. Within 48 hours after feeding, normoxic and anemic pythons exhibited heart rates 78% and 126% higher, respectively, than fasted controls. While no PCH was found in fed normoxic animals, fed anemic animals exhibited 38% more massive hearts. Similar cardiovascular responses and hypertrophies occurred in fed anemic alligators. These findings support our hypothesis that an oxygen supply‐demand mismatch is the upstream stimulant of PCH, a phenomenon now known to exist across at least two taxa of reptiles. Funding provided by the Danish Research Council to TW and NSF grant IOS 0922756 to JWH. CES received support from an NSF GRF and a SICB FGST.
A dynamic model of the hypoxia‐inducible factor (HIF) network
Authors: Cheong Alex , Cavadas Miguel AS , Nguyen Lan , Scholz Carsten C , Fitzpatrick Susan F , Bruning Ulrike , Tambuwala Murtaza , Manresa Mario C , Kholodenko Boris N , Taylor Cormac C
Published: June 2021

Activation of the hypoxia‐inducible factor (HIF) pathway is a critical step in the transcriptional response to hypoxia. While many of the key proteins involved have been characterised, the dynamics of their interactions in generating this response remain unclear. We have generated a comprehensive mathematical model of the HIF pathway based on core validated components and dynamic experimental data. Our model confirms that the steps leading to optimal HIF transcriptional activity require sequential inhibition of both prolyl‐and asparaginyl‐hydroxylases. We also predict from our model and show experimentally that there is residual activity of the asparaginyl‐hydroxylase FIH at low oxygen tension. Furthermore silencing FIH under conditions where prolyl‐hydroxylases are inhibited results in increased HIF transcriptional activity but paradoxically decreased in stability. Using a core module of the HIF network and mathematical proof supported by experimental data, we propose that asparaginyl hydroxylation confers resistance upon HIF to proteosomal degradation. Thus, through in vitro experimental data and in silico predictions, we provide a comprehensive model of the dynamic regulation of HIF transcriptional activity by hydroxylases and use its predictive and adaptive properties to explain counter‐intuitive biological observations.
Prolyl Hydroxylase 1 (PHD1) and Factor Inhibiting HIF (FIH) regulate IL‐1β‐induced NF‐κB activity linking key hypoxic and inflammatory signaling pathways
Authors: Scholz Carsten C , von Kriegsheim Alexander , Tambuwala Murtaza M , Hams Emily , Cheong Alex , Bruning Ulrike , Fallon Padraic G , Cummins Eoin P , Taylor Cormac T
Published: June 2021

Low oxygen concentration (hypoxia) is a feature of chronically inflamed tissues. Hydroxylases are oxygen‐sensing enzymes, which control the transcriptional response to hypoxia and influence the course of inflammation through the regulation of HIF‐and NF‐κB‐dependent pathways. Four different oxygen‐sensing hydroxylases are known: Prolyl Hydroxylase 1, 2 and 3 (PHD1, 2, 3) and Factor Inhibiting HIF (FIH). Pharmacologic hydroxylase inhibition reduces inflammation in animal models of colitis, reperfusion injury and sepsis, however, the underlying mechanisms remain unclear. IL‐1β is a major pro‐inflammatory cytokine that activates NF‐κB‐dependent transcriptional pathways and is associated with numerous inflammatory pathologies. We investigated a role for hydroxylases in regulating IL‐1β‐dependent inflammatory signaling. We show that hydroxylase inhibition reduces IL‐1β‐induced NF‐κB activity in a manner, which is dependent upon the combinatorial inhibition of PHD1 and FIH and results in repression of NF‐κB‐dependent genes. Hydroxylase inhibition reduced IL‐1β‐induced signaling upstream of JNK, p38 and IKK. Thus, combinatorial inhibition of PHD1 and FIH represents a new approach to the inhibition of inflammatory signaling pathways activated by IL‐1β, which may be of benefit in inflammatory disorders.
Effect of Beta Adrenergic Stimulation and Blockade on Intrapulmonary Arteriovenous Anastamoses Recruitment
Authors: Bates Melissa , Jacobson Joseph , Hotter Jessica , Eldridge Marlowe
Published: June 2021

Intrapulmonary arteriovenous anastomoses (IPAVs) are large diameter conduits that bypass the pulmonary circulation. IPAVs can be recruited in humans by catecholamines and it has been suggested that this occurs as a result of an elevated cardiac output. In contrast, we hypothesized that IPAVs recruitment occurs as a result of beta adrenergic‐mediated vasodilation. To test our hypothesis, we measured the transpulmonary passage of 2.5×105 venous‐injected microspheres (25–50 μm) in anesthetized, room air ventilated rats infused with either saline (n=6) or isoproterenol (n=6) and found substantial microsphere passage in the isoproterenol group (0.4±0.9% vs 10.5±15.2%, p=0.007). However, because the contributions of cardiac output and vasodilation cannot be isolated in the intact model, we also perfused isolated rat lungs with buffered hetastarch (n=6) or isoproterenol (n=9), taking care to match pulmonary artery pressure and flow between groups. Transpulmonary microsphere passage occurred only in isoproterenol perfused lungs (1±3 vs 200±278, p=0.001). This suggests that, contrary to previous conclusions, catecholamines recruit IPAVs independently of increases in cardiac output, through active vasodilation. In subsequent preliminary work, we hypothesized that catecholamines are necessary for full IPAVs recruitment by hypoxia. In rats ventilated with 10% O2, pre‐treatment with propranolol (n=4) decreased the passage of microspheres by 58% compared to controls (n=5) (14.7±25.0 vs 6.1±3.2, p=0.10) but did not abolish the response. This suggests that the response to hypoxia is multifactorial, but that catecholamines are important contributors to IPAVs recruitment in hypoxia.
Unraveling premature breathing and apneas of prematurity in mice
Authors: Koschnitzky Jenna E , Smith Charles V , Ramirez Jan Marino
Published: June 2021

We used lipopolysaccharide (LPS) to induce preterm birth in mice and then characterized breathing using both plethysmography and in vitro brainstem slices containing the preBötzinger Complex (preBötC). LPS was injected (5μg i.p.) at gestational day 17.5 (G17.5). Labor commenced on G18.5, one day early. The pups fell into one of three categories: stillborn, died soon after birth, or survived. Of the pups that died soon after birth, the breathing patterns consisted of large, very infrequent inspirations. Pups that survived went through a progression of breathing patterns which resulted in normal breathing within 60 minutes. In vitro extracellular recordings of the preBötC in pups with breathing patterns indicative of imminent death showed slow, irregular burst patterns while pups that breathed normally had burst patterns similar to control mice born at G19.5. In these pups, daily plethysmograph recordings showed that the occurrence of apneas ceased at G20.5 in LPS pups compared to G21.5 in control pups (P2 in all animals). Control pups were also compared to control pups that failed to thrive (FtT) at P1. The FtT group never achieved normal weight and had severe apneas that continued until P5. These deficits do not appear to be associated with abnormal preBötC function. The results indicate that preBötC function is associated with survival of preterm mice while apneas associated with FtT are independent of the preBötC.
Central Expression of IL‐1β and Cardioventilatory Uncoupling in a Rodent Model of Sepsis
Authors: Hsieh Yee‐Hsee , Nethery David , Jacono Frank , Dick Thomas
Published: June 2021

Sepsis is an overwhelming systemic infection that leads to cardio‐respiratory failure and is associated with the loss of heart rate variability (HRV). The mechanisms by which the immune response to sepsis affects HRV, in particular cardio‐respiratory coupling (CRC), are unclear. We hypothesize that expression of pro‐inflammatory cytokines in the nucleus tractus solitarius (nTS) disrupts CRC. To begin to test this hypothesis, adult male Sprague Dawley rats (n=6) were anesthetized with isoflurane, and diaphragmatic EMG and ECG were recorded. After a 30‐min ‘baseline’ recording, lipopolysaccharide (LPS) (24 mg/kg) or saline was injected intraperitoneally. After 6h, respiratory and heart rates increased relative to baseline in LPS‐ but not saline‐treated rats. Coefficient of variation did not change in either group. Immunostaining revealed upregulation of IL‐1β in nTS. Cardioventilatory coupling was assessed by chi‐squared analysis of single‐ordered histograms. After LPS injection, chi‐squared value decreased from 71.5 ± 0.92 to 36.6 ± 12.3 (mean ± SD, p<0.01). We conclude that IL‐1β expression in the nTS is associated with cardiorespiratory uncoupling in sepsis. These data support the concept that CRC, in addition to HRV, could serve as a biomarker for health status and affects gas‐exchange efficiency contributing to cardio‐respiratory function.
Neonatal programming of the ventilatory control system: effects of inflammation and fasting
Authors: Shirahata Machiko , Kostuk Eric , Mason Ariel , Pichard Luis , Kesavan Kalpashri , Tankersley Clarke , Gauda Estelle
Published: June 2021

Perinatal environmental stress has long‐term effects on many organs. In the carotid body (CB), hyperoxia during perinatal periods impairs development and results in attenuated responses to hypoxia in adulthood. Because the CB is a multimodal sensor, we hypothesized that more common insults, inflammation or under‐nutrition, at early postnatal life has prolonged effects on CB function. In the group 1 (Sprague Dawley rats), lipopolysaccharide (LPS, 50–100 μg/kg, IP) was given at P2. CB output from the whole carotid sinus nerve showed that hypoxic response was attenuated in LPS‐treated animals at P9–10 and ~P30. In the group 2, DBA/2J mouse pups (P5–13) were fasted for 3 or 6 hours in a warm cage (~33 °C). Fasting reduced body weight (<5%) and blood glucose. Ventilation was measured with plethysmography in sleeping pups. After fasting, baseline breathing was slower and sporadic. Hypoxia increased ventilation, but the distribution of breath intervals was shifted toward slower breathing vs control. During hyperoxia, breath intervals distributed widely with prolonged apneas and no peaks. These features persisted 1 day after the pups were fed and gained weight. Five days later, the baseline breathing was still slower than control, but apnea numbers decreased. These results support the hypothesis. Inhibited function of the CB may be, at least partly, responsible for the disturbed ventilation. (HL81345, R25DA021630)
Characterisation of H441 cells as an in vitro model of distal lung epithelial barrier
Authors: Ehrhardt Carsten , Gausterer Julia Clara , Schwagerus Elena , Salomon Johanna Jessica
Published: June 2021

No continuously growing cell line of human distal lung epithelial phenotype and with the ability to form polarised, electrically tight monolayers has been described to date. Here, we investigated, if the human lung adenocarcinoma cell line H441 has potential to overcome this issue.

Epithelial barrier properties were studied by immunocytochemistry (ICC) against ZO‐1 and measurement of RT. The expression of MDR1 and organic cation/carnitine transporters (OCT/Ns) was also investigated by ICC and immunoblot. Uptake of [14C]‐TEA and [3H]‐acetylcarnitine was carried out to assess OCT/N function. Furthermore, the effects of OCT/N modulators on organic cation uptake were studied.

H441 cells formed confluent, electrically tight monolayers with peak RT values of 600–1000 Ω·cm2, after 8 days in culture. Presence of P‐gp, OCT1–3, OCTN1 and 2 was confirmed by Western blot and ICC. Uptake of organic cations was time and temperature‐dependent. In the case of [14C]‐TEA, verapamil and MPP+ most efficiently inhibited uptake, whilst amantadine and HC‐3 did not show an effect. D‐carnitine and ergothioneine significantly attenuated [3H]‐acetylcarnitine uptake into H441 cells.

H441 cells have the ability to form monolayers with appreciable barrier properties. Moreover, their drug transporter expression and activity is consistent with what has been reported for human alveolar epithelial cells in primary culture.
MicroRNA expression regulates cardiac fibrosis in Uremic Cardiomyopathy
Authors: Drummond Christopher Anson , Haller Steven , Shapiro Joseph I , Tian Jiang
Published: June 2021

In rats, uremic cardiomyopathy via 5/6th partial nephrectomy (PNx) displays cardiac hypertrophy and fibrosis. Studies in our lab have revealed a crucial role for Na/K‐ATPase signaling as mediated through cardiotonic steroids in the development of this phenotype. Molecular regulators of this disease process are poorly understood; as such we found reports linking fibrotic diseases with micro RNA (miRNA) expression. Reports indicate that miR‐29b directly targets collagen mRNA and that expression of miR‐29b is decreased in fibrotic tissues. We examined miR‐29b levels in rat hearts from the following groups: sham controls, PNx and MBG‐infused animals. The qPCR data showed that PNx and MBG‐infusion, significantly decreased miR‐29b by 3.18‐ and 2.33‐fold, respectively. In point of fact, collagen expression increased significantly in both PNx (7.7‐fold) and MBG‐infused (6.4‐fold) animals versus sham controls. Additional array based studies revealed that in PNx and MBG‐infused animals 17 miRNAs were dysregulated in both groups these miRNAs include: 29b, 124, 144, 122, 32, 141,196b, 96, 423, 210, 375, 182, 196a, and 324–5p. These miRNAs were found to be associated heavily with the development of fibrosis and apoptosis. We conclude that dysregulation of miRNA expression is mechanistically linked with the development of the phenotype observed in uremic cardiomyopathy. Supported by NIH HL‐105649
Activity dependent regulation of Na,K‐ATPase α3 mobility in the postsynaptic membrane
Authors: Kruusmägi Markus , Liebmann Thomas , Brismar Hjalmar , Aperia Anita
Published: June 2021

The rapid changes in intracellular Na+ concentration due to excitatory synaptic activity by AMPA receptors are offset by the Na,K‐ATPase which is responsible for restoring the membrane potential and for maintaining a low intracellular level of sodium. Many recent studies describe the dynamics of AMPA receptor diffusion which should consequently modulate the influx of Na+. Though regulation of AMPA receptors and Na,K‐ATPase are likely coordinated, a direct link has never been investigated. In this study we use live cell imaging methods to examine population and single molecule movement of the Na,K‐ATPase a3 isoform and explore a functional regulation associated with AMPA receptor activity. Using Super Ecliptic pHluorin‐tagged al3 and FRAP we measured population mobility in cultured hippocampal neurons. We found that a majority of the Na,K‐ATPase molecules in the dendritic membrane are in lateral diffusion. With quantum dot labeling, we measured the single molecule diffusion in the extrasynaptic and synaptic membranes. After AMPA receptor stimulation the mobility of a3 was significantly increased in both the extrasynaptic and synaptic membranes. Using TTX to inhibit neuronal activity we observed a decreased mobility in the extrasynaptic and synaptic membranes. The alterations seen in response to changes in synaptic activity demonstrate that a3 is functionally regulated in the dendritic membrane.
A specific and essential role for Na,K‐ATPase α3 in neurons co‐expressing α1 and α3
Authors: Kruusmägi Markus , Azarias Guillaume , Connor Siobhan , Akkuratov Evgeny E , Brismar Hjalmar , Broberger Christian , Aperia Anita
Published: June 2021

Most neurons co‐express two catalytic isoforms of Na,K‐ATPase, the ubiquitous α1, and the more selectively expressed α3. Although neurological syndromes are associated with α3 mutations, the specific role of this isoform is not completely understood. Here we used electrophysiology and sodium imaging to study the role of α3 in neurons expressing both isoforms. Under basal conditions, selective inhibition of α3 using a low concentration of the cardiac glycoside ouabain resulted in a modest increase in the intracellular sodium concentration ([Na+]i), accompanied by membrane potential depolarization. When neurons were challenged with a large rapid increase in [Na+]i, similar to what could be expected following suprathreshold neuronal activity, inhibition of α3 almost completely abolished the capacity to restore [Na+]i in dendrites. Recordings of Na,K‐ATPase‐specific current supported the notion that α3 is the predominant isoform, responsible for rapid extrusion of Na+. Low concentrations of ouabain were also found to disrupt cortical network oscillations, providing further support for the importance of α3 function in the central nervous system. The α isoforms express a well conserved protein kinase A (PKA) consensus site, which is structurally associated with a Na+ binding site. Activation of PKA significantly attenuated both the α3 dependent current and restoration of dendritic [Na+]i, indicating that α3 is a target for phosphorylation and may participate in short‐term regulation of neuronal function.
Decreased Activation of the X‐linked inhibitor of Apoptosis Protein (XIAP) is Associated with Increased Apoptosis in the Preeclamptic Placenta
Authors: Price Matthew Dean , Arroyo Juan Alberto
Published: June 2021

Normal placentation and placental development are critical for a successful pregnancy and mediate important steps such as implantation and delivery of nutrients to the fetus. Preeclampsia (PE) is characterized by an increase in placental apoptosis, however the mechanism is not conclusive. The X‐linked inhibitor of apoptosis protein (XIAP) protects against apoptosis. Our objective was to determine the level and localization of active XIAP protein in PE placenta compared to controls. Control and PE placental samples were collected shortly after delivery. Tissues were frozen in liquid Nitrogen for RNA and western blot studies. For immunohistochemical studies tissues were fixed and embedded in paraffin and stained for activated (phospho) XIAP. We observed: 1) a trend for a significant decrease in XIAP gene expression (60%) in the PE placenta as compared to controls; 2) No significant differences in XIAP protein expression; 3) a significant decreased in active (phospho) XIAP protein (30%; p<0.05) in the placentas of PE women; and 4) decreased in active (phopspho) XIAP immunostaining in the syncitiotrophoblast layer of the PE placenta. Our data confirms that XIAP protein is expressed by the syncytiotrophoblasts in the human placenta during PE. We conclude that the decrease in activation of this protein during PE could potentially account for the increased apoptosis observed in this tissue at term. This result gives insights into pathways associated with the increased placental apoptosis and could be useful in the understanding of potential mechanisms that lead to placental insufficiency associated with this disease.
Anti‐proliferative effect of ethylacetate fraction from Orostachys japonicus on modulation of MAPK signaling pathways in human gastric cancer cells
Authors: Ryu Deok‐Seon , Kim Seon‐Hee , Kwon Ji‐Hye , Lee Dong‐Seok
Published: June 2021

This study focused on the anti‐proliferative effect of ethylacetate (EtOAc) fraction of the herbal plant Orostachys japonicus. This study examined the effect of EtOAc fraction on human gastric cancer (AGS) cell proliferation and investigated the underlying mitogen‐activated protein kinases (MAPK) signaling pathways. The EtOAc fraction inhibited the proliferation of AGS cells by inducing cell cycle arrest as evidenced by propidium iodide (PI) labeling and RT‐PCR analysis. In the cell cycle analyses, treatment with the EtOAc fraction resulted in a marked increase of cells in the sub‐G1 and G2/M phases. RT‐PCR was performed to study the correlation between G2/M arrest and transcription of cell cycle control genes. The anti‐proliferative effect of EtOAc fraction was accompanied by inhibition of CDK1 and cyclin B1 mRNA. To understand the role of MAPK signaling in the anti‐proliferative effect of EtOAc fraction in AGS cells this study assessed EtOAc fraction‐treated AGS cells using antibodies against p38 MAPK (p38), c‐Jun N‐terminal kinase (JNK), and extracellular signal‐regulated kinase (ERK). MAPK such as p38, JNK, and ERK were analyzed by Western blotting. Treatment of AGS cells with the EtOAc fraction resulted in time‐ and dose‐dependent activation of p38, JNK, and ERK.
Expression profiles of three isoforms of phospholipase C Xdomain containing protein (PLCXD) in human and mouse
Authors: Kalujnaia Svetlana , Gellatly Steven , Cramb Gordon
Published: June 2021

Phosphatidylinositol‐specific phospholipase C (PI‐PLC) enzymes are a family of receptor‐regulated phosphodiesterases that control many cellular processes by the regulation of cytosolic calcium and/or the activity of several protein kinases. Recently we identified a seventh class of PI‐PLC enzymes (called PLC‐XDs), which contains only the catalytic X domain of the normal multi‐domain‐ containing enzyme family. Three tissue‐specific PLC‐XD isoforms exist in humans, hPLC‐XD1, ‐XD2 and ‐XD3, with ‐XD2 exhibiting three C‐terminal spliceforms. Similar PLC‐XD orthologs have been identified in mice with the mPLC‐XD3 having two C‐terminal spliceforms. PLC‐XD isoforms exhibited their own individual tissue‐specific expression profiles and sub‐cellular localisations with some similarities between species. PLC‐XD1 and ‐XD3 isoforms were expressed in most tissues tested with PLC‐XD2 mainly found in leucocytes and thymus in humans, but predominately in the GI‐tract and stomach of mice. PLC‐XD3, which exhibits the highest sequence homology across species, was mainly expressed in mouse and human heart. The expression profiles suggest that these novel proteins may possess fundamental, and as yet uncharacterised tissue‐specific roles in cell physiology.

Funding: School of Medicine, University of St Andrews
Molecular Determinants of TRPC Channels Gating by STIM1
Authors: Yuan Joseph P , Lee Kyupil , Graham Sarabeth , Ahuja Malini , Ma Rong , Muallem Shmuel
Published: June 2021

We investigated if STIM1 SOAR, which has a coiled‐coil (CC) domain, interacts with the TRPC channel N‐ (NT) and/or C‐terminal (CT) CCs by determining how mutations in the TRPC CCs affect their interaction with SOAR and their gating by STIM1. We found that SOAR binds directly to TRPC1, TRPC4 and TRPC5, but not to TRPC3 and TRPC6. Mutations of 3 residues in the NT CCs of all TRPCs eliminate their surface expression and reduce binding of TRPC1 and TRPC5 to STIM1, while increasing binding of TRPC3 and TRPC6 to STIM1. In contrast, most single mutations in the NT CC of TRPC3 enhance binding to STIM1 with no appreciable effect on surface expression. Notably, I807S in the CT CC of TRPC3 has no effect on its interaction with STIM1, but eliminates the increased interaction observed by mutations of the NT CC. Accordingly, sensitivity of TRPC3 and TRPC3(L245S) to STIM1 knockdown by siRNA and scavenging STIM1 by the channel dead Orai1(R91W) show that a greater inhibition of STIM1 function is required to inhibit the function of TRPC3(L245S) compared to WT TRPC3. Lastly, TRPC3(I807S) eliminates regulation of TRPC3 and TRPC3(L245S) by STIM1. These findings suggest a model in which the TRPC3 NT and CT CCs interact to shield the CT CC from binding to STIM1. Dissociation of this interaction allows STIM1 SOAR to access and bind to the CT CC, to present the STIM1 polybasic domain to the conserved negative charges on TRPCs, and, ultimately, to open the channel.
Pregnant rats maintained on sodium overload shows reduced type‐1 VEGF receptor in placenta
Authors: Vieira‐Filho Leucio Duarte , Ribeiro Natalie Emanulle , Gonçalves Daianna Rosse Martins , Borges Luís Paulo Nogueira Cabral , Cabral Edjair Vicente , Soares Anísio Francisco , Paixão Ana Durce Oliveira
Published: June 2021

Sodium overload during pregnancy compromises the renal function of offspring at adult life. In this work we investigated whether maternal sodium overload affects placental lipid peroxidation and type‐1 VEGF receptor (VEGFR‐1) in the placenta. Pregnant Wistar rats were maintained with sodium chloride 1.8%, instead drinking water, from 20 days before pregnancy until the 20th day of pregnancy. Part of them was treated with α‐tocopherol or tempol from the 1st day of pregnancy. Maternal sodium overload did not change the placental or hepatic fetal lipid peroxidation; however it decreased the expression of VEGFR‐1 in placental labyrinth. Treatment with α‐tocopherol or tempol prevented sodium overload‐induced reduction in placental expression of VEGFR‐1. Different from tempol, α‐tocopherol effect was accompanied by reduction of lipid peroxidation in maternal placenta and fetal liver. These findings indicate that intrauterine programmed renal dysfunction produced by sodium overload might be due to compromised placental angiogenesis that is not correlated to placental lipid peroxidation. CAPES, CNPq and FACEPE.
Apelin regulation of potassium chloride cotransport (KCC) in vascular smooth muscle cells (VSMCs): Relation to Cardiovascular Disease (CVD)
Authors: Sharma Neelima , Lauf Peter K , Adragna Norma C
Published: June 2021

Apelin, a potent inodilator, uses the NO pathway to elicit its anti‐atherogenic effect, and the PI3K/Akt and MAPK pathways to induce proliferation and migration of VSMCs. The NO pathway is predominant in healthy blood vessels and contractile VSMCs, whereas, the PI3K/Akt/MAPK pathways are prominent in diseased blood vessels and secretory VSMCs. VSMCs participate in atherosclerotic lesions due to their capacity to migrate and proliferate. Oxidized plasma cholesterol (oxLDL) is a detrimental factor in atheroma formation. It is known that KCC uses the same regulatory pathways (Adragna et al. 2006) as apelin and is implicated in CVD. Here, we tested the hypothesis that apelin through its APJ receptor signaling pathways regulates KCC. Expression and distribution patterns of contractile proteins were studied by Western blot and immunofluorescence. KCC activity was measured by atomic absorption spectrophotometry with Rb as K congener ± inhibitors of the aforementioned signaling pathways and by blocking other K+ transport mechanisms. The APJ receptor and key components of the signaling pathways were verified immunologically. VSMCs’ identity was established by appropriate markers. Apelin stimulated KCC activity through the NO pathway by 335 % and by 142 % through the MAPK/PI3K pathways, In contrast, oxLDL inhibited baseline KCC in contractile VSMCs, and this inhibition was restored by apelin. These findings prove the hypothesis that apelin regulates KCC activity through the aforementioned pathways, are relevant to CVD and have potential as therapeutic targets. Supported by WSU Foundation.
Increased MMP7 expression in the aging kidney causes upregulation of collagen
Authors: Slusarz Anna , Nichols LaNita A , Grunz‐Borgmann Elizabeth A , Parrish Alan R
Published: June 2021

MMP7 is the smallest member of the metalloproteinase family and has gained attention in the recent years for its role in abnormal tissue remodeling. The secreted protein is minimally expressed in the adult except for a few niche tissues such as the small intestine and bladder. However, MMP7 is reactivated in a host of disease states including cancer and chronic kidney disease. We have found a dramatic increase in MMP7 expression in aged rat kidneys, with significant elevation starting at 16 months, and over 500 fold up‐regulation in 2 year‐old animals compared to 4 month old rats. Interestingly, 2 year‐old animals, which were maintained on a calorie‐restricted diet, had MMP7 expression comparable to 4 month‐old controls.

In order to delineate the contributions of MMP7 to renal dysfunction, we overexpressed full‐length pro‐form of MMP7, which is not processed to a catalytically active form in vitro, a constitutively catalytically active mutant MMP7, and an inactive mutant in the normal rat kidney cell line NRK‐52E. High‐throughput sequencing of the cells revealed significant changes in expression of two collagen alpha genes, Col1a2 and Col3a1, which contribute to fibrosis.

Importantly, both of these genes were up‐regulated in aged rat kidneys and the increased expression temporally correlated with increased MMP7 expression and fibrosis.

This study was supported by NIH AG034145.
Impact of summer research experiences on educational & career paths of rural & tribal undergraduate students
Authors: Doze Van A , Conway Pat , Olimb Sarah , Wages Jennifer , Sens Don , Cisek Karen
Published: June 2021

Undergraduate years are critical for engaging students in research, to increase the number pursuing STEM careers. Research opportunities at home institutions are sparse for rural and tribal college students, underrepresented in science. Through our NSF‐funded Research Experience in Neuroscience for Students from Rural and Tribal Colleges (REU), eight student cohorts have completed a 10‐week summer research experience. The past four summers, other institutional resources were leveraged to add additional students into our REU program. We utilized student feedback to enhance the summer experience and determine student outcomes, measured using pre‐ and post‐surveys and focus groups. Students report high impact in terms of strengthening lab skills and increased scientific literacy and self‐confidence. We assessed the influence of the research experience and other factors, personal, family, and community, on student career choices. A significant number of rural and tribal students have progressed to post‐baccalaureate programs. Analyzing these factors has allowed a better understanding of the contribution of undergraduate research experiences to post‐undergraduate study and career choices. Supported by National Science Foundation REU Site grants (0639227, 0851869) and by grants from the National Institutes of Health National Center for Research Resources (P20 RR016471) and the National Institute of General Medical Sciences (P20 GM103442).
Sympathetic renal nerve ultrastructure: Comparative analysis between adult mice and rats.
Authors: Carvalho Carolina Silva , Sato Karina Laurenti , Castania Jacy Airton , Nessler Randy Alan , Salgado Helio Cesar , Fazan Valéria Paula Sassoli
Published: June 2021

The renal nerves exhibit efferent and afferent sympathetic fibers, crucial for regulation of renal function and input of sensory information from the kidneys to the central nervous system. The intrinsic renal innervation was widely described in rats. However, few reports deal with renal extrinsic innervation in rats and mice, and comparative studies between species have not been reported. We aimed to compare the ultrastructural and morphometric characteristics of the extrinsic renal nerves of adult rats and mice. A total of 15 left renal nerves, 5 from adult male C57BL/6 mice and 10 from adult male Wistar rats were used. The renal nerves were dissected and prepared for light and electron microscopy morphometry. As expected, the difference of the fascicular area and diameter was significant, being the nerves of mice smaller than rats. The myelinated fibers of rats were larger (diameter and area) compared to mice, but the number and density of these fibers were not different between species. No morphological differences were observed (number, area, diameter, and size distribution) on the unmyelinated fibers between species. Our results indicate that fascicle and myelinated fibers sizes accompany the animal size. The unmyelinated fibers, mainly responsible for the sympathetic functions of the renal nerve, do not differ between species in size and number, suggesting the absence of functional differences.
Investigation of Arterial Vascularization of Kidney and Adrenal Gland in White Laboratory Mice; Acroanatomic Subgros and Scanning Electron Microscopic Study
Authors: Alan Aydýn
Published: June 2021

Laboratuary mice are valuable species that are commonly used in different studies. The physiology and anatomy of related organs and tissues must be well known to have clinical practices, sugical operations and organ transplantations. Although the most commonly used in scientific research laboratory mice living things no research on the kidney and adrenal gland heve been performed. When we planned this study and found no such studies in the literature rewiew. For this purpose examined the arterial vascularization of kidney and adrenal gland of 24 units white Balb‐c laboratory mice. We obtained different magnifications of images both on corrosion casts and live tissues by dissection with latex injection, corrosion cast and electron microscopy techniques. The kidneys were determined to be supplied by A. renalis which gives of from aorta as a sigle branch. No anatomosis were observed in the branching arteries of kidneys but the polar arteries were observed in both left and right kidneys. Gl. suprarenalis dextra was spherical where Left adrenal gland was drop like in shape. A. suprarenalis cranialis, a. suprarenalis media and a. suprarenalis caudales were observed to supply left adrenal gland. Out of 24 materials, only one had right adrenal gland supplied by a. suprarenales media whereas in the remaining materials only a. suprarenalis caudalis were supplying right adrenal gland. Besides none of the materials were observed to have a. suprarenalis cranialis.
Serum VEGF as a marker of endometrial receptivity in infertile women
Authors: Paul Shipra , Jain Pooja , Tuli Anita , Gupta Usha , Jain Manjula
Published: June 2021

Purpose

Female infertility is multifactorial in origin and may be a reflection of suboptimal endometrial receptivity. Vascularity is an essential pre‐requisite for continuation of pregnancy. Vascular endothelial growth factor (VEGF) is a major regulator of endothelial cell proliferation, angiogenesis, vasculogenesis, and capillary permeability. We assessed the effect of serum VEGF levels on grades of endometrial receptivity.

Methods

60 infertile women ages 20–40 years, were enrolled in the study after local IRB approval. Time of ovulation was determined by TVS Doppler ultrasonography just prior to premenstrual biopsy for endometrial characterization and sub‐endometrial blood flow. Grading of endometrial receptivity was based on histopathological findings during mid‐luteal phase. The serum VEGF levels were estimated by ELISA.

Results

Serum VEGF levels increased with grades of endometrial receptivity: 38.48±10.60 pg/ml in Grade I, 86.75±17.25 pg/ml in Grade II, and 215.08±25.60 pg/ml in Grade III. The difference of mean serum VEGF level between grades of endometrial receptivity was highly significant p=0.001.

Conclusions

Serum VEGF level of 87 pg/ml predicted adequate endometrial receptivity with a sensitivity of 89.5% and a specificity of 87.8%.
Telocytes and stem cells in regenerative medicine
Authors: Nicolescu Mihnea Ioan , Manole Catalin Gabriel
Published: June 2021

Introduction

A newly described type of interstitial cells – the telocytes – takes part in the repair/regenerative processes in several organs, including adult mammalian heart. Telocytes form a complex interstitial network, establishing hetero‐cellular contacts with resident stem/progenitor cells.

Methods

Immunohistochemistry, transmission electron microscopy and cellular electron tomography were used to identify and characterize the presence and intercellular relations of telocytes in rat heart (n=10) and human salivary glands (n=5). Experimental myocardial infarction was induced and samples from different time intervals (24h, 48h, 1 week) were compared with normal hearts.

Results

Cardiac telocytes play an essential role in “nursing” progenitor cells in cardiac stem cell niches. The putative salivary stem cells are also in close contact with telocytes prolongations (telopodes), creating a specific microenvironment, in several periacinar or periductal areas.

Conclusion

Pathology of both vital (heart) and non‐vital (salivary glands) organs may lead to mild or severe dysfunction(s). The deciphering of local tissue repair and renewal mechanisms is of utmost significance. Telocytes may have the potential to increase the efficiency of self‐healing capacity, in a close tandem with resident stem cells.

Grant Funding Source: AAA
Expression of stem cell markers in beta cell of the human pancreas
Authors: Filgueira Luis , Baashar Ahmaed
Published: June 2021

The purpose of this study was to assess the expression of embryonic stem cell and pancreas‐specific markers in insulin producing beta cells in adult human islets, including Nanog, Oct4, Sox2, menin, Ngn3 and PDX‐1.

An indirect immune fluorescence staining was done on archival formalin‐fixed, paraffin‐embedded human pancreatic samples. Expression of the three beta cells precursor markers menin, Ngn3 and PDX‐1 were identified in the islets, always co‐localized with insulin. Expression of Nanog, Oct4 and Sox2 were distinctly identified in the islets of the human pancreas. Oct4 and Sox2 expression was always co‐localized with insulin, indicating that all insulin secreting beta cells do express these two markers. Nanog was mainly co‐expressed with insulin in beta cells, but there was a small number of cells in the islets expressing Nanog, but no insulin, demonstrating that Nanog might be expressed by the others endocrine cells or early precursors or tissue‐specific stem cells.

These findings demonstrate that the three embryonic stem cell markers Nanog, Oct4 and Sox2 are expressed in the islets of the human pancreas. The precursor markers of beta cells such as menin, Ngn3 and PDX‐1 are always localized with insulin producing beta cells as well. Consequently, the presence of embryonic stem cell markers in the human adult pancreas may offer a new principle for regenerating and replacing ageing or damaged beta cells.
Host pathogen interactions: new lessons from Listeria monocytogenes
Authors: Cossart Pascale
Published: June 2021

Listeria monocytogenes is a bacterial pathogen that hijacks different host cell components at different stages of the infection. The talk will focus on the invasion process which involves two receptors E‐cadherin and the receptor tyrosine kinase Met. Interactions of the bacteria with the receptors activate a series of molecular events that lead to entry. We will show in particular how we discovered the unexpected key role of clathrin in the actin rearrangements used for entry, what are the main actors involved in clathrin recruitment and in the connection of clathrin to actin. A complete picture is emerging from RNAi screens which will be discussed. In addition, we will show how the study of Listeria entry into cells has helped discovering a so far not described role for clathrin in cell cell junctions formation.
Gene Gap Investigation of Chalcone Synthase in California Tarweeds and the Hawaiian Silverswords
Authors: Myers Tiffany Leigh , Dias Kristen , Vandergon Virginia
Published: June 2021

Complex organisms such as plants have many different metabolic pathways that influence substrate specific reactions and when changed can lead to speciation events. The anthocyanin biosynthetic pathway (ABP), which is a part of the Phenylpropanoid pathway, controls many different mechanisms that regulate temperature, UV protection, microbial invasion, fruit, seed and flower coloring. In the ABP, Chalcone synthase (CHS) is an enzyme that is responsible for catalyzing the first condensation reaction and essential for other genes further down the pathway to function. The model system we are using is the Hawaiian Silverswords, which are of great interest due to the fact that they are a classic model of adaptive radiation and have large phenotypic but little genotypic differences. In previous studies copies of CHS in Dubautia scabra, Dubautia linearis, and Wilkesia gymnoxiphium in the Hawaiian Silverswords as well as Madia elegans, Madia gracilis, and Holocarpha macradenia, which are apart of the California tarweeds (Silversword Progenitor), have been partially sequenced, analyzed and identified as CHS. The gaps that remain include exon 1 and 2 for Wilkesia gymnoxiphium, and Dubautia linearis as well as the intron region which will help in distinguishing specific gene copies within each genome. We accomplish this by using polymerase chain reaction, cloning, sequencing, and bioinformatics.
Characterization Of CD‐209 Isoforms With Carbohydrate Recognition Domain Modified Due To Alternative Splicing
Authors: Santos Luciana Lara , Pereira Lailah Horácio Sales , Taranto Alex Gutteres , Junior Moacyr Comar , Lopes Débora Oliveira , Siqueira Jaqueline Maria
Published: June 2021

The CD‐209 gene encodes a glycoprotein expressed as soluble or membrane, mainly present in human macrophages and dendritic cells. It is responsible for initial adhesion of the dengue vírus (DENV) to these cells. This gene undergoes alternative splicing, which results in thirteen different transcript of isoforms and consequently of the glycoprotein. The aim of this study is to predict the molecular structure of CD‐209 isoforms with carbohydrate recognition domain (CRD) modified due process of splicing alternative. The isoforms were obtained from the GenBank and aligned by Multialin software. Analysis in silico was performed to verify possible differences in CRD. 3D model structures were built by homology modeling (Swiss‐Model Server). We have demonstrated three sites of Ca2+ bind in CRD and amino acids residues that take part of it. We have found five isoforms with different CDR and only two of them keep three sites of Ca2+ bind. Validation steps were done and root‐mean‐square deviation values were found below 0.6. These variations may be correlated to the pathogenesis of dengue, since the virus needs of these sites of Ca2+ bind to interact with CD‐209 receptor. If the isoforms which miss some sites of Ca2+ are frequent in some populations this could confer a certain protection against virus infection. Future population studies would be interesting to better understand these issues.

Support: FAPEMIG
Influence of butyrate, a histone deacetylase inhibitor, on expression profile of microRNAs in vascular smooth muscle cells (VSMC)
Authors: Ranganna Kasturi , Mathew Omana P , Milton Shirlette G
Published: June 2021

Butyrate, a dietary‐derived epigenetic histone modifier and a histone deacetylase inhibitor, is an inhibitor of VSMC proliferation, a critical element in vascular remodeling and restenosis. Butyrate elicits cytoprotective, chemopreventive and chemotherapeutic activities through inhibition of cell proliferation, induction of cell death and/or cell differentiation by modulating gene expression via epigenetic changes. Because butyrate is an established epigenetic histone modifier, expression of certain microRNAs (miRNAs) may be altered by butyrate via epigenetic mechanisms including DNA methylation and histone modification. To explore this possibility, we examined expression profile of 650 miRNAs in butyrate inhibited rat VSMC proliferation by qRT‐PCR array platform. Our results indicate differential expression of 60 miRNAs. Among these, members of miRNA‐17–92 cluster are some of the miRNAs that are downregulated by butyrate in VSMC. Considering the role of miRNA‐17–92 cluster in oncogenesis and overexpression in different cancers, underexpression of members of miRNA‐17–92 cluster in butyrate arrested VSMC suggests antiproliferation action of butyrate is linked to corresponding increase in target genes of miRNA‐17–92 cluster, such as CDKN1A (p21Cip1). Overall, our miRNA expression data emphasizes role of miRNAs in antiproliferative and chemoprotective effects of butyrate in VSMC.
Use of Pseudomonas aeruginosa Chaperones in Folding an Insect Recombinant Prolyl Endoprotease (rPEP) Expressed in Escherichia coli.
Authors: Gautam Ranjeeta , Dareddy Vanitha , Clack Beatrice
Published: June 2021

Chaperone assist in protein folding. The purpose of this study were to 1) determine if co‐expression of Pseudomonas aeruginosa GroESL or Trigger factor with prolyl endo protease (PEP) cDNA in Escherichia coli system resulted in increase amount of soluble active enzyme 2) determine if two different temperatures, 37°C for 24 hours and biphasic temperature incubation of 37°C for 6 hours followed by overnight in 28°C would increase in amount of soluble active PEP and 3) to identify which chaperone mechanism results in highest expression of active protein. GroESL and TF chaperones were cloned into pLys‐S and pLys‐E plasmid. The resulting pGROELS‐S/E plasmid or pTF‐S/E plasmid was co‐transformed into E. coli. Western Blot techniques were used to determine the level of protein expression. Enzyme activity was assayed using a synthetic chromagenic peptide. Biphasic temperature growth resulted in higher level of active than constant 37°C. pGroESL‐ E and pTF‐S resulted in highest increase in amount of soluble rPEP as compared to amount found. Expression in presence of pGroESL‐E and pTF‐S resulted in the highest levels of active soluble enzyme. P. aeruginosa GroESL and TF chaperones co‐express and assist in folding successfully expression of a large insect PEP in E. coli.

Funding: Office of Research and Sponsored Projects, Stephen F Austin State University; Ed and Gwen Cole, Nacogdoches, TX.
Understanding K11‐ polyubiquitin recognition at the 26S Proteasome
Authors: Chaney Joseph , Das Chittaranjan
Published: June 2021

Ubiquitination, the covalent attachment of ubiquitin to target proteins, is a reversible post‐translational modification that regulates many important cellular processes. Ubiquitin chains linked at Lys48 (K48) serve as a signal for proteasomal degradation; whereas K63‐linked chains have non‐proteolytic function. Lysine 11 (K11)‐linked chains are upregulated when the human E3 anaphase‐promoting complex (APC/C) is activated during mitosis, which initiates substrate degradation by attaching K11‐linked chains for proteasomal targeting. However, why cells use K11‐linked chains instead of K48 for this purpose and how the chains are recognized by the shuttle factors and receptors on the proteasome are not known. At the proteasome it has been well established that there are two ubiquitin receptors that preferentially bind K48‐linked chains, RPN10 and RPN13. We are investigating the role of hRpn13 conserved pleckstrin‐like receptor domain (Pru) and the hRpn10 ubiquitin interacting motifs (UIM) in binding to K11‐linked di‐ubiquitin. We show that K11‐linked di‐ubiquitin binds Rpn13Pru domain with similar affinity as K48 and K63‐linked di‐ubiquitin. These interactions may play an important role in cell cycle regulation by serving as the recognition site for proteolytic degradation and implies that the proteasome may use other ubiquitin binding domains to discriminate between different chain types.