bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostaglandin D2‐DP1 receptor signaling inhibits sympathetic nerve activity and development of angiotensin II‐salt hypertension (874.11)
Authors: Asirvatham‐Jeyaraj Ninitha , Fink Gregory
Published: June 2021

Cerebrospinal fluid levels of lipocalin‐prostaglandin D synthase protein (L‐PGDS) and the lipid mediator prostaglandin D2 (PGD2) are increased in early stages of AngII‐salt hypertension (AngII‐HS‐HTN) in rats. Because L‐PGDS inhibition reduces neurogenic AngII‐HS‐HTN development and its product PGD2 acts on central DP‐1 receptors (DP‐1Rs), we hypothesized that PGD2 acting through DP1‐Rs mediated AngII‐HS‐HTN development.

Method: Two sets of studies were performed. First, anesthetized rats received an injection of PGD2 (50µg/5µl) intracerebroventricularly (icv). PGD2 caused a maximum 22±6 mmHg increase in BP. In other anesthetized rats the DP1‐R antagonist BW A868C (78ng/5µl) was injected icv followed by PGD2 injection; BW A868C blocked PGD2 mediated increases in BP. In the second study we tested the effect of chronic DP1‐R blockade on AngII‐salt HTN. Continuous radio‐telemetric recording of BP was used. After control BP recording, rats on 2% NaCl diet were pretreated with vehicle (n=4) or BW A868C (425nM/h, sc, n=5). All rats then received AngII (150ng/kg/min, sc) for 14 days. Ganglionic blockade with hexamethonium (30mg/kg, ip) was performed on day 10 of AngII administration to assess neurogenic pressor activity. Finally, PGD2 (50µg/5µl) was injected acutely into lateral ventricles to test if central DP‐1Rs were inhibited.

Result: Compared to the control period, AngII infusion increased mean arterial pressure (MAP, mmHg) from 95±2 to143±10 (31±10 increase) in vehicle, and from 109±7 to140±1 (56±3 increase) in BW A868C treated rats (p<0.05). Neurogenic pressor activity (mmHg) was significantly higher in BW A868 (86±10) than vehicle (46±11) treated rats. Finally, central PGD2 injection failed to elicit a BP response in BW A868C treated rats confirming central DP1‐R inhibition.

Conclusion: Acute icv PGD2 induced increases in BP are mediated via DP‐1R. However, chronic PGD2 signaling through central DP1‐R inhibits AngII‐HS‐HTN development and causes sympathoinhibition.

Grant Funding Source: “Supported by” RO1 HL 076312 and RCO HL 060363
Examining egg laying circadian rhythm phenotypes in a glycolytic mutant fly model (883.3)
Authors: Hrizo Stacy , Finver Ethan , Hollingsworth Rachel , Stonebraker Jonathan , Yaccobucci Kathryn
Published: June 2021

Triosephosphate Isomerase (TPI) is a glycolytic enzyme. Mutations in this enzyme are associated with a neurodegenerative disorder called TPI Deficiency in humans. In addition to neurological health other cellular processes may be affected by alterations in sugar metabolism. We developed an experiment to assess if flies with a mutant TPI allele called “sugarkill” exhibit reduced egg production or alterations in the circadian rhythm of egg production. Egg production is a highly energy dependent process and therefore alterations in energy production may decrease the ability of the animals to produce eggs. In addition, flies exhibit circadian rhythm phenotypes, of which, include egg laying primarily in the morning. If neurological function is altered, the circadian rhythm for the flies may be altered as well. For this experiment, two different genotypes of Drosophila melanogaster were utilized: flies were either homozygous for the wildtype TPI allele or the mutant TPIsugarkill allele. With each genotype matings were set up with 4 virgin females to every 12 males and then the number of eggs was counted every 4 hours during 12 hour cycles from 7am‐7pm in an incubator set at 25C. Results indicate that while the average clutch size is not different during a 24 hour period the circadian rhythm pattern for egg laying and eclosion is altered in TPIsugarkill flies.
Dietary quercetin enrichment improves respiratory function in mdx mice (884.17)
Authors: Selsby Joshua , Ballman Christopher , Quindry John
Published: June 2021

Duchenne muscular dystrophy (DMD) is caused by a dystrophin deficiency and results in progressive muscle injury culminating in death due to respiratory or cardiac failure. We have established that gene transfer of the exercise mimetic, PGC‐1α, will decrease disease severity and improve muscle function in skeletal muscle taken from mdx mice, the mouse model of DMD. While this approach is effective and repeatable, at present it is not readily translatable. Hence, the purpose of this project was to determine the extent to which oral delivery of quercetin, a PGC‐1α activator, would improve respiratory function in mdx mice. To address this purpose baseline respiratory function was measured in vivo in two month old C57 and mdx mice. After measurement, the mdx group was randomly assigned into groups receiving a control diet (mdx) or diet enriched with 0.2% quercetin (mdx‐Q). Respiratory function was measured again at four and six months of age. By six months of age we found that a 16% (p<0.05) increase in respiratory frequency and a 12% (p<0.05) increase in tidal volume caused a near 30% (p<0.05) increase in minute ventilation in mdx‐Q compared to mdx. Further, peak inspiratory flow was increased 20% (p<0.05) and peak expiratory flow was increased 26% (p<0.05) in mdx‐Q compared to mdx. These data suggest strongly that dietary enrichment of the orally available PGC‐1α activator, quercetin, attenuated muscle injury and improved respiratory function in a mouse model of Duchenne muscular dystrophy.

Grant Funding Source: Supported by the Duchenne Alliance and its member foundations
Oxygen tension regulates H2S and NO bioavailability in reciprocal manner (885.3)
Authors: Kevil Christopher , Shen Xinggui , Shiva Shruti , Gladwin Mark , Roach Robert
Published: June 2021

Nitric oxide and hydrogen sulfide modulate several physiological and cellular functions including vasodilation, cell proliferation and redox cell signaling. Production and bioavailability of either molecule may be influenced by oxygen tension; however, the relationship between H2S and NO bioavailability during hypoxia remains poorly understood. In this study, we examined plasma free H2S and nitrite levels in healthy volunteers exposed to high altitude. Plasma free H2S levels were significantly greater at sea level compared to high altitude (5260 meters) at 1 or 16 days; whereas, plasma nitrite levels were significantly higher at high altitude compared to sea level. After spending 7 or 21 days at low altitude, volunteers were tested again at 5260 m. On reascent, plasma free H2S levels were increased compared to initial high altitude exposure. Conversely, plasma nitrite levels were lower than high altitude exposure on reascent indicating reciprocal regulation between the gasotransmitters. Calculation of plasma free H2S to nitrite ratios revealed that high altitude acclimatization decreases the ratio of plasma free H2S to nitrite, and that on reascent H2S to nitrite ratios return to low altitude levels. In vitro cellular hypoxia studies using HUVEC revealed significant decreases in acid labile sulfide while increasing protein bound biochemical forms of sulfide. Interestingly, in vitro hypoxic challenge similarly decreased free H2S to nitrite ratios in HUVEC similar to high altitude exposure. These data demonstrate a unique reciprocal relationship between H2S and NO bioavailability during hypoxia.
Telemetry‐based recording of renal cortical oxygenation in conscious rats with chronic kidney disease (890.4)
Authors: Koeners Maarten , Emans Tonja , Ow Connie , Evans Roger , Malpas Simon
Published: June 2021

Multiple forms of acute and chronic kidney disease are associated with renal tissue hypoxia. This has led to the proposition that renal hypoxia is not just a consequence of kidney disease, but rather a primary pathogenic event. Using telemetric technology to chronically measure the partial pressure of oxygen (PO2), we recorded cortical PO2 during the early stages of development of chronic kidney disease (CKD) in conscious rats. Male Wistar rats underwent 5/6 subtotal nephrectomy (n = 4) or sham (n = 4) surgery. After an initial increase, cortical PO2 values decreased in the first 2 weeks after induction of CKD and thereafter remained stable for the subsequent 6 weeks with an average reduction of 42 ± 12% compared to baseline. Sham operation had little effect on cortical PO2. The decrease in tissue PO2 in the remnant kidney occurred before proteinuria and uremia suggesting that hypoxia precedes the symptoms of CKD and therefore could be a major driver of the subsequent pathology. This is consistent with the hypothesis that kidney hypoxia is central in the pathogenesis of CKD.

Grant Funding Source: EU, FP7, Marie Curie Actions, International Outgoing Fellowship
Dietary NaCl modulates the urinary organic metabolite profile: influence of the organic anion transporter OAT3 (892.34)
Authors: Vallon Volker , Fu Yiling , Kuczkowski Alexander , Anzai Naohiko , Nigam Sanjay , Eraly Satish , Gerasimova Maria , Ramachandra Rao Satish
Published: June 2021

The organic anion transporter 3 (OAT3) mediates uptake of organic anions across the basolateral membrane of the proximal tubule in exchange for α‐ketoglutarate. Mice lacking OAT3 have reduced blood pressure, but the underlying mechanism is unclear. Renal metabolism is closely linked to transport function, but little is known about the metabolic response of the kidney to changes in NaCl diet. Here we show that a low NaCl diet (0.01%) increases renal OAT3 protein expression by 50‐60% vs. high NaCl diet (4%) and that NaCl intake has distinct effects on the urinary metabolite pattern in wild‐type mice (WT): α‐ketoisocaproate, α‐ketoisovalerate, α‐keto‐3methyl valerate, and α‐ketoglutarate were not detectable in urine under high NaCl diet, whereas a low NaCl diet was associated with mean urinary concentrations of 50‐300 ng/µmol creatinine (each P<10‐6). Low NaCl diet was also associated with 10‐20 fold higher urinary excretion of pyruvate (P<10‐3). In contrast, urinary excretion of benzoate, 3‐OH‐butyrate, malonate, and succinate were lower under low vs. high NaCl diet (each P<10‐3), the latter being also an activator of GPR91, which stimulates renin release. Similar differences were observed in Oat3‐/‐ mice, except that a consistent and significant stimulatory effect of low NaCl diet on urinary α‐ketoglutarate was not detected. We conclude that dietary NaCl intake has a distinct influence on the urinary organic metabolite pattern, including substrates and components of the citric acid cycle and other molecules used as energy source. This pattern is mainly independent of OAT3, even though dietary NaCl regulates renal expression of OAT3.

Grant Funding Source: Supported by NIH grants R01HL94728, R01DK56248, P30DK079337, and the Department of Veterans Affairs
A druggable binding site in the emerging diuretic target ROMK (893.15)
Authors: Swale Daniel , Sheehan Jonathan , Meiler Jens , Denton Jerod
Published: June 2021

An emerging body of evidence suggests the Renal Outer Medullary potassium channel (ROMK, Kir1.1) represents a novel diuretic target for the treatment of hypertension. We recently employed high‐throughput screening and medicinal chemistry to develop the first highly selective small‐molecule ROMK inhibitor, VU591 (Bhave et al., 2011). Its clean ancillary pharmacology raises important questions about how drug selectivity can be achieved in a structurally simple K+ channel. The voltage‐dependence of VU591 block suggests the binding site is located in the membrane‐spanning pore, prompting us to perform scanning mutagenesis analysis of this region of ROMK. Most mutations had no effect on VU591 sensitivity; however, mutation of Val168 and Asp171, which are unique to ROMK among other inward rectifier K+ channels, led to side chain‐specific changes in channel block. In support of our functional data, comparative molecular modeling and in silico docking analysis revealed an energetically favorable interaction between VU591 and the side chains of V168 and N171. This study reveals key structural elements required for selective inhibition of this emerging diuretic target.

Grant Funding Source: Supported by NIH 1RO1DK082884 to Jerod S Denton
Modulation of KCa3.1 by ubiquitylation in a polarized epithelium (893.20)
Authors: Lee Shih‐Liang , Devor Daniel , Hamilton Kirk
Published: June 2021

In this study, we examined the role of ubiquitylation in the retrograde pathway of KCa3.1, for the first time in a polarized epithelium. Previously, we have shown that UBEI‐41, an inhibitor of ubiquitinin‐activating enzyme, reduced ubiquitylation and internalization of KCa3.1 (Balut et al. FASEB J 25: 2938‐48, 2011) in a non‐polarized epithelium. We asked the question ‘Is KCa3.1 ubiquitylated at the basolateral membrane?’ In order to test this, we used our Fischer Thyroid epithelial cell line stably expressing KCa3.1‐BLAP along with Bir‐A‐KDEL (cells grown on filters) that allows the biotinylation of channels prior to leaving the Golgi. Initially, we determined the time course of KCa3.1 by labeling (at 4C) the surface channels with streptavidin and then incubating cells at 37C for 0, 1, 3, 5, 8 and 12 hours. The half‐life of KCa3.1 was 3 hrs (n=6). To confirm that KCa3.1 was ubiquityated in a polarized epithelium, we conducted a similar experiment, as above, in which the cells were incubated with UBEI‐41 (50 µM). UBEI‐41 increased the half‐life of KCa3.1 to 12 hrs (n=4). If, channels were ubiquitylated at the membrane, in the presence of UBE‐41, then we hypothesized that there would be an increase number of channels at the membrane. To test this hypothesis, we used the Ussing chamber to examine an increase in function of KCa3.1 in the presence of UBEI‐41. Indeed, after 1 hr, K current in the presence of UBEI‐41 was increased compared to the K current of cells that were not treated with UBEI‐41. These data suggest that KCa3.1 is ubiquitylated at the membrane prior to being internalized.

Grant Funding Source: This work was supported by the Department of Physiology, Univ. of Otago (KLH) and NIH (DCD).
Identification of two extracellular sites in the transmembrane region of SLC26A9 chloride channel ő Cl/HCO3 exchanger (893.27)
Authors: Chen An‐Ping , Chang Min‐Hwang , Romero Michael
Published: June 2021

The SLC26A9 protein mediates Cl‐ transport as a Cl‐ channel and a Cl‐/HCO3 exchanger in the several epithelial cells, e.g., gut, lung, kidney. Topology analysis from BicA, a member of the SulP/SLC26A family, suggests that Slc26 proteins consists of 12 predicted transmembrane regions. However, the topology model of human SLC26 transporters has not yet been directly investigated. In this study, 12 HA‐inserted SLC26A9 mutants were constructed and expressed in Xenopus oocytes. Chemiluminescence was used to determine which HA‐tags were accessible from the extracellular surface. 157‐160 and 352‐353 sites of human SLC26A9 were identified as extracellular sites. Interestingly, the SLC26A9 HA (159‐160) mutant has ~40% Cl‐ transport activity, but this decreased function could be rescued by coexpression with another low function cSNP (T127N‐SLC26A9). These results revealed that the topology of TM region of SLC26A9 may different from that of BicA and also indicate that human SLC26A9 functions as dimer.

Grant Funding Source: Supported by NIH EY 017732 (MFR)
Na+/H+ exchanger NHE9 affects tumor progression of human glioblastomas by altering endosomal pH (893.34)
Authors: Llongueras Jose , Kondapalli Kalyan , Hack Anniesha , Capilla‐González Vivian , Smith Chris , Guerrero‐Cazares Hugo , Quiñones‐Hinojosa Alfredo , Rao Rajini
Published: June 2021

Na+(K+)/H+ Exchangers (NHEs) are a large family of transport proteins that conduct electroneutral countertransport of cations with protons across lipid bilayers. A newly discovered Na+/H+ Exchanger, NHE9 (SLC9A9), has recently been shown to localize to a perinuclear region corresponding to recycling endosomes, where it is thought to regulate luminal pH and control cargo trafficking and degradation. Given the mounting evidence implicating NHE9 in neurological diseases including autism and ADHD, we explored the functional consequence of altered NHE9 expression levels in brain cancer.

Glioblastoma Multiforme (GBM) is the most aggressive form of malignant glioma that accounts for over 50% of all gliomas and is frequently characterized by the amplification and membrane persistence of epidermal growth factor receptor (EGFR). EGFR signaling is tightly regulated by receptor endocytosis and lysosomal‐mediated degradation, and we postulated that NHE9 might alter this pathway. Here, we have found that levels of NHE9 are amplified in a large subset of GBMs. Using primary GBM cell cultures with naturally high or low levels of NHE9, we have found that altering those levels regulates tumorigencity and migratory capacity in vitro and in nude mouse xenografts. Furthermore, increased NHE9 levels alkalinizes the luminal pH of endosomes, which inhibits EGFR degradation and promotes oncogenic signaling downstream of MAPK and Akt. Finally, we show that inhibiting NHE9 with amiloride‐derived inhibitor EIPA increases EGFR responsiveness to Erlotinib. Our findings demonstrate that NHE9 is a target gene that should be explored as a treatment for a subset of GBMs.

Grant Funding Source: Supported by NIH grant R01 DK054214
Conserved polarization of Na,K‐ATPase in vertebrate skin epithelia (895.1)
Authors: Modyanov NIikolai , Korneenko Tatyana , Shakhparonov Mikhail , Pestov Nikolay
Published: June 2021

Development of epidermis creates stratified epithelium with different sets of ion‐transporting enzymes in its layers. We have characterized expression of Na,K‐ and H,K‐ATPases (X=Na or H) in rat skin including all α and β subunits and six FXYD isoforms. Na,K‐ATPase is predominantly represented by α1 and β3 isoforms. Among H,K‐ATPases, only the α‐subunit of non‐gastric H,K‐ATPase (αng) was detected. Expression of ATP1A1, ATP1B4 and FXYD6 genes are down‐regulated in adult rat skin as compared to the newborn one, whereas the level of αng and FXYD4/CHIF are higher in adult skin. Immunohistochemistry with the use of monoclonal antibodies indicates that αng and α1 are differentially expressed during keratinization: α1 is gradually decreased whereas αng is enriched in the outer live cell layer, stratum granulosum. Na,K‐ATPase α1 is abundant in cells of the innermost layer, stratum basale, and, unexpectedly, is completely undetectable in their basal membranes. Epidermis of a frog species, Xenopus laevis, also show lateroapical location of Na,K‐ATPase, as well as in the chicken epidermis. These findings indicate that Na,K‐ATPase in epidermis is not basolateral as it is in most epithelial cells. On the other hand, Na,K‐ATPase is basolateral in epithelium of the cutaneous secretory glands. These features appear to be conserved in tetrapod vertebrates.Supported by RFBR grant 13‐04‐01413.

Grant Funding Source: RFBR grant 13‐04‐01413
High saturated fat diet alters skeletal muscle phospholipid composition and increases SERCA activity (895.2)
Authors: Trojanowski Natalie , Castelli Laura , Miotto Paula , Ward Wendy , LeBlanc Paul
Published: June 2021

Sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) is a transmembrane protein whose activity is, in part, dependent on its lipid environment. Past studies using synthetic membranes have demonstrated that 20‐22 carbon long monounsaturated fatty acids are associated with maximal SERCA activity. However, this association has not been examined in biological membranes. Thus, the objective of this study was to examine this association in a biological system and the response to high saturated fat (HSF) diet‐mediated membrane alterations in skeletal muscle. Female Wistar rats (28 days old) were fed control (CON; AIN93G, 7% soybean oil by weight) or HSF (AIN93G, 20% lard by weight) diet for 16 weeks. Mixed hind limb muscle phospholipids from rats fed a HSF diet had lower percent mole fraction of polyunsaturated fatty acids, unsaturation index (UI), and average chain length (ACL); and higher saturated fatty acids and SERCA activity compared to CON (p<0.05). Correlation analysis demonstrated ACL (R2= 0.83, p < 0.05) and UI (R2=0.53, p = 0.062) were negatively correlated with SERCA activity. These results suggest that SERCA activity may be influenced by phospholipid composition in biological membranes differently than in synthetic membranes. Future studies will examine the lipid annulus closely associated with SERCA.

Grant Funding Source: Supported by Natural Sciences and Engineering Research Council (NSERC)
Amino acids involved in lubiprostone activation of human ClC‐2 (896.11)
Authors: Cuppoletti John , Malinowska Danuta , Chakrabarti Jayati , Tewari Kirti , Ueno Ryuji
Published: June 2021

Objective. The study’s objective was to determine amino acids important for activation of human ClC‐2 (hClC‐2) by lubiprostone. NMR studies of ClC‐0 by Alioth et al (J Mol Biol 369:1163‐9, 2007) identified an α‐helical region that interacts with ClC‐0 CBS domains, important to channel regulation, which contains one of two PKA phosphorylation sites unique to hClC‐2 (RGET691).

Methods. Whole cell patch clamp of wild type and mutant hClC‐2 expressed in EBNA293 cells were carried out as described (Cuppoletti et al, Cell Biochem Biophys 66: 53‐63, 2013). Wild type rabbit and mouse ClC‐2 were also studied.

Results. Single amino acid mutagenesis of the PKA phosphorylation site RGET691‐>A shifted the EC50 for lubiprostone stimulation of wild type hClC‐2 from 27.6 ± 4.5 nM (4) to 445.8 ± 2.1 nM (3). The triple mutant RGET691‐>AGAA lost all lubiprostone stimulation. Both mutants were still stimulated by forskolin/IBMX. Both rabbit and mouse ClC‐2 were also stimulated by lubiprostone with EC50s of 350 ± 37.9 nM (3) and 64.3 ± 3.7 nM (4), respectively.

Conclusions. RGET691 plays an essential role in lubiprostone stimulation of human ClC‐2. In light of NMR evidence that this region may interact with CBS domains and contains the RGET691 phosphorylation site in hClC‐2, binding of lubiprostone to this region in human, rabbit and mouse ClC‐2 may alter the interaction of this region with CBS domains, leading to direct modulation of ClC‐2.

Grant Funding Source: Supported by Sucampo AG
Cdc42 plays a requisite role in dopamine transporter endocytic trafficking (896.6)
Authors: Wu Sijia , Melikian Haley
Published: June 2021

Dopaminergic neurotransmission is essential for voluntary movement, reward and cognition, and dopamine (DA) signaling is implicated in Parkinson’s disease, addiction and schizophrenia. The presynaptic Na+/Cl‐‐dependent DA transporter (DAT) recaptures released DA, thereby temporally and spatially sculpting DAergic neurotransmission. DAT is potently inhibited by addictive (cocaine, amphetamine [AMPH]) and therapeutic (methylphenidate) psychostimulants. DAT loss‐of‐function mutations lead to infantile Parkinsonism and several DAT coding variants are associated with ADHD. Thus, intrinsic cellular mechanisms modulating DAT availability are likewise predicted to significantly impact DAergic neurotransmission. PKC activation and AMPH exposure acutely decrease DAT surface expression via robust endocytic trafficking. However, the mechanisms governing DAT membrane trafficking are not clearly defined. We recently demonstrated that 1) Rin GTPase activity is required for PKC‐stimulated DAT internalization and 2) DAT endocytic recycling relies on a dynamin/actin‐dependent mechanism. Both Rin and the actin cytoskeleton are intimately coupled to cdc42, suggesting that cdc42 activity may impart multimodal control over DAT endocytic trafficking. Here, we used small molecule cdc42 inhibitors and cdc42 mutants to test this possibility in dopaminergic cells lines and acute mouse striatal slices. Cdc42 inhibitors casin and pirl1 significantly decreased DA uptake in SK‐N‐MC cells. DAT functional losses were accompanied by decreased DAT surface expression in SK‐N‐MC cells and in mouse striatal slices. Studies using shRNA and cdc42 GTPase mutants will further define the role of cdc42 in DAT membrane trafficking.

Grant Funding Source: DA035244
The orphan drug carbaglu is a substrate of the sodium dicarboxylate transporter 3 (896.8)
Authors: Burckhardt Birgitta , Schwob Elisabeth , Hagos Yohannes , Burckhardt Gerhard
Published: June 2021

Inborn defects in the carbamoylphosphate synthase 1 (CPS1) cause a reduction of N‐acetylglutamate (NAG), an essential cofactor for the function of the urea cycle, and consequently elevated blood levels of the neurotoxic ammonia. N‐carbamoylglutamate (carglumic acid, carbaglu®, NCG) is a structural analog of NAG that can substitute for NAG on CPS1, thereby reactivating the urea cycle and finally reducing blood ammonia levels. The renal clearance of NCG exceeds the glomerular filtration rate suggesting an active secretion process in kidney proximal tubules. The kidneys are known to express organic anion transporting polypeptides (OATPs) and organic anion transporters (OATs). OATs work in cooperation with sodium‐dicarboxylate transporters (NaDCs) thereby mediating the exit of many drugs. The impact of NCG on several human OATPs, OATs, and NaDCs stably transfected in HEK293 cells was tested. From the transporters tested, OAT1, OATP1B3, and NaDC3 interacted with NCG. Sodium‐ and concentration‐dependent NCG‐mediated currents were measured in Xenopus laevis oocytes expressing NaDC3. NCG was identified as a high affinity substrate of NaDC3 with a KM of 27.5 µM. This value equals the plasma concentration of succinate, the lead substance of NaDC3 and as well as the plasma concentration of patients treated with NCG. Competition of succinate and NCG on NaDC3 may result in reduced renal clearance of drugs.
Investigation of MCT1 transporter protein in the human gastrointestinal tract (897.1)
Authors: Al‐mousawi Hashmiyah , Courtney Danielle , McGrane Alison , Winter Desmond , Baird Alan , Stewart Gavin
Published: June 2021

Bacterially‐derived short chain fatty acids, such as butyrate, are vital in maintaining the symbiotic relationship that exists between humans and their gastrointestinal microbial populations. A key step in this process is the uptake of butyrate into colonic epithelial cells, via MCT1 transporters, which regulates cell differentiation (Cuff et al., 2005). Although previously studied, the exact localisation of MCT1 protein along the human gastrointestinal tract remains controversial (Gill et al., 2006; Iwanaga et al., 2006). The aim of this study was therefore to re‐investigate MCT1 protein abundance in various human gastrointestinal tissues. Initial RT‐PCR analysis confirmed the expected MCT1 RNA expression pattern of colon > small intestine > stomach. Using surgical resection samples, Western analysis utilising the MCT12A antibody (Source Bioscience, UK) detected higher abundance of a 45 kDa MCT1 protein in colon compared to ileum (P<0.05, N = 4, unpaired t‐test). Importantly, MCT1 abundance was found to be significantly higher in ascending and descending colon compared to sigmoid colon (P<0.01, N = 11, ANOVA). In contrast, ileum samples obtained from ileostomy reversal surgeries showed a high MCT1 abundance, with no significant difference between proximal and distal ileum (NS, N = 7, paired t‐test). These data appear to confirm that MCT1 protein abundance is correlated to levels of bacterially‐derived short‐chain fatty acids along the gastrointestinal tract. In addition, they highlight the importance of precise tissue location in studies comparing colonic MCT1 abundance between normal and diseased states.

Grant Funding Source: Supported by Science Foundation Ireland and the Kuwaiti government
MCT‐1 and SMCT‐1 regulation by miR‐29a, b and c in intestinal epithelial cells (908.8)
Authors: Anbazhagan Arivarasu , Borthakur Alip , Alrefai Waddah , Dudeja Pradeep
Published: June 2021

Monocarboxylate transporter‐1 (MCT1) and sodium‐coupled monocarboxylate transporter‐1(SMCT‐1) are the major proteins involved in the absorption of short chain fatty acids (SCFA) in the colon. SCFAs exert myriad of beneficial effects including the amelioration of inflammation. Therefore, it is important to understand the mechanisms of regulation of MCT1 and SMCT‐1 expression in health and disease. MicroRNAs (miRs) are known to play a key role in modulating gene expression. In this regard, miR‐29 a, b have been recently shown to alter MCT1 expression in pancreatic ‐cells. Whether these miRs modulate colonic MCT1 and SMCT1expression is not known. The 3`‐untranslated regions (UTR) of MCT1 and SMCT‐1 genes were cloned into pmirGLO vector upstream of luciferase and transienstly transfected with miR29a,b and c mimics into colonic Caco‐2 and T‐84 cells. Luciferase activity and MCT1 and SMCT‐1 expression were determined. The presence of UTR of these genes significantly decreased luciferase activity (~40%) as compared to pmiRGLO empty vector. Co‐transfection with miR‐29 a, b or c resulted in further decrease in 3`‐UTR activity of MCT1 and SMCT1 luciferase constructs. MCT1 and SMCT‐1 mRNA expression was not altered in response to mimic transfections, whereas the protein expression was significantly decreased (~30%). Further, the expression of miR 29 a and c was significantly lower in mouse colon as compared to small intestine, consistent with higher levels of MCT1 and SMCT1 protein in the colon. Our studies demonstrated that miR 29 a, b and c down‐regulate MCT1 and SMCT‐1 expression in colonic epithelial cells and may partly explain the differential expression of these transpoters along the length of the GI tract.

(Supported by NIDDK and Dept of Veteran Affairs)

Grant Funding Source: NIDDK and Dept of Veteran Affairs
Geometric morphometrics of the distal radioulnar joint of felids and canids (914.11)
Authors: Carlon Burcu
Published: June 2021

The shapes of the articular surfaces of the distal radioulnar joint were analyzed through geometric morphometric techniques in felids and canids. The species analyzed include A. jubatus, N. nebulosa, F. catus, V. vulpes, S. fatalis, P. atrox, and C. dirus. The objective was to demonstrate the supination and pronation capabilities through comparing the differences in the shapes of the articular surfaces. Landmarks were chosen around the facet for radius on distal ulna and facet for ulna on distal radius. The results of the Generalized Procrustes and Principal Components analyses had A. jubatus, a cursorial felid, cluster with the canids V. vulpes, and C. dirus and differ significantly from the felids in the analysis. For the ulna, PC1 accounted for 93.9% and PC2 for 2.2%. For the radius, PC1 made up 90% and PC2 2.1%. The wireframe depictions of the articular surfaces are elongated proximodistally in A. jubatus and the canids. In the felids the articular surfaces are expanded in the anteroposterior direction instead. A. jubatus and canid forearms are locked in the prone position with less rotation available than in the felids. This analysis appears to be related to the differences in the locomotor behaviors of species and may prove to be useful in comparing cursorial and arboreal species.
Specialize or disappear: anisomerism, empirical evidence of the differentiation or loss of polyisomere structures during gnathostome evolution based on a comparative study of head and appendicular muscles (918.27)
Authors: Ziermann Janine , Diogo Rui
Published: June 2021

Some authors have defended the notion of anisomerism, i.e. that there is an evolutionary tendency for the original polyisomeres of an organism (e.g., branchial arches, digits) to become differentiated so that phylogenetically derived organisms do not consist of a linear series of similar parts. This notion has been often related with the notion of functional uncouplings, i.e. that the differentiation of a polyisomere into an anatomically different structure allows this structure to not only have a new function but also to be liberated from its original functional complex and therefore to become functionally associated with other structures of the body. We provide here the first detailed empirical analysis of this issue based on data obtained from a comparison of the head and appendicular muscles of all major groups of gnathostomes. Our results support the idea that there is effectively a trend for the muscles associated for instance with the branchial arches or with the digits to specialize or, instead, to become lost in evolution. One example concerns our own species, Homo sapiens, which almost completely lacks any of the original similar polyisomere head muscles found in taxa such as sharks (e.g., branchial adductors), and that lacks many of the original similar polyisomere autopodial (hand/foot) muscles found in tetrapods such as salamanders or lizards (e.g., the flexores breves superficiales).

Grant Funding Source: Faculty Start‐Up Package, Howard Univ. Coll. Medicine
Creating testing regimes for terrestrial origins of the avian flight stroke (919.8)
Authors: Dececchi Thomas , Habib Michael , Hall Justin
Published: June 2021

While the ancestry of birds has been firmly established within maniraptoran theropod dinosaurs, the exact timing of the origins of the flight stroke remains elusive. Debate rages as to the drivers of this critical step in the evolution of powered flight, but little biomechanical modeling has been done to test the various hypothesis proposed. Here we seek to test three proposed pathways for a terrestrial origin of flight: flap running, Wing Assisted incline Running (W.A.I.R.) and burst leaping, to examine where, if at all, on the non‐avian to avian theropod tree the aerodynamic forces required by each are possible. Based on figures generated for in‐vivo experimental data from the literature we find that W.A.I.R. is restricted, conservatively, to the sister clade to birds while flap running is biomechanically unlikely. These results will help to shape future discussion on the ecology and drivers of the origin of flight by placing theses behavioral possibilities in their evolutionary context.
Avascularity in the cortical bone of small bats as revealed by micro computed tomography (920.3)
Authors: Pratt Isaac , Walker Ernest , Cooper David
Published: June 2021

The spatial distribution of vascular canals in cortical bone reflects the mechanical loading conditions present during the formation of the bone. In the flight bones of birds, these canals have a laminar pattern, oriented circumferentially around the medullary cavity. Evidence for this pattern and its relation to torsional loading has thus far come from histological studies, however vascular canals exist in 3D and their cross sections in a 2D slice may vary from their actual 3D morphology. We hypothesized that bats would show the same laminar pattern in their flight bones and that high resolution 3D imaging would better reveal the extent of the cortical network. We used micro‐computed tomography (micro‐CT) to examine the cortical network of two species of bats (Eptesicus fuscus pallidus, Lasiurus cinereus), two species of birds (Tyrannus verticalis, Seiurus aurocapillus), and two species of terrestrial mammals (Mus musculus, Eutamias minimus), all of similar size. Contrary to our expectations we found that the bats had cortices devoid of vascular canals. Compared to the terrestrial mammals, we found that the birds had a reduction in the number of cortical vascular canals present and did not exhibit the anticipated laminar pattern found previously in larger bird species.
Effects of insertion forces on the surface wear of dental implants (922.14)
Authors: Adapalli Arvind , Sridhar Sathyanarayanan , Thomas Anie , Valderrama Pilar , Wilson Thomas , Rodrigues Danieli
Published: June 2021

The main goal of this study was to understand the effects of insertion forces on the wear properties of dental implants. It is hypothesized in this study that titanium dental implants may be subjected to wear during implantation, which can result in particle generation and accumulation in the surrounding tissues. Metal particle accumulation may contribute to adverse tissue reaction and diseases like peri‐implantitis. To quantify particle generation upon implant insertion, 4 dental implants (Straumann USA LLC, 10 mm) were inserted in sawbone blocks of increasing densities, 10 PCF, 20 PCF, 30 PCF, and 40 PCF (pounds per cubic feet, PCF), simulating implantation in the bone. After insertion, each implant was removed from the block, which were sectioned for microscopic inspection. The internal surface of the sectioned blocks, the surface of the implants, and the compositions of the particles were analyzed with 3D microscopy (Keyence, VHX200), Scanning Electron Microscopy (JEOL 5600), and powder X‐ray (Bruker D8 Advance). 3D microscopy and Scanning Electron Microscopy revealed the presence of metal debris deposited in each density of sawbone . The amount of particles deposited was observed to decrease with increasing sawbone density. Powder X‐Ray Diffraction confirmed that particles generated were mainly composed of titanium. Therefore, this study illustrates how the insertion of dental implants may lead to the generation of metal particles in the encompassing tissue. This suggests that deformation of the implant surface and particle accumulation may start to develop in stages as early as insertion.

Grant Funding Source: University of Texas at Dallas Startup Funds (Dr. Rodrigues)
Instrumentation for capture of high‐speed anatomical movement (923.1)
Authors: Sochor Mark , Heltzel Sara
Published: June 2021

Objective: Describe the technique of instrumentation of the human body for capturing anatomic motion at high speed. A human body interacting with its environment is at risk for injury. Anatomic structures with viscoelastic properties do not behave in the same way at high speed. It's necessary to study these interactions when developing mechanical surrogates of the human body (crash test dummies). Way in which a crash dummy is able to replicate a human’s actual movement is called biofidelity.

Methods: PMHS in this series were fresh frozen specimens thawed to room temperature. Instrumentation techniques which incorporate hardware into the spine and ribcage of the PMHS will be described. Video capture of these PMHS and the acceleration data were captured and compared in an automotive crash environment. Acceleration data filtered at industry standard and photo target tracking system compared to the post‐processed acceleration data. Force defection curves generated as well as excursion measurements for the head, thoracic spine, pelvis and lower extremities.

Results: The PMHS moves similar to a live human subject in a high G environment as muscle reaction time is minimized and the baseline muscle tone is often overcome. Excursion of the PMHS head, thoracic spine and pelvis over several milliseconds shows the importance of anatomical movement in an acceleration environment.

Conclusions: Instrumentation techniques have been developed. Techniques and motion capture are highly accurate and aid in developing surrogate tools for the motion of the human body for safety product development. Although PMHS do not have the baseline muscle tone of a live subject they do have realistic soft tissue strength (ligaments, tendons and other soft tissue load bearing structures). Accepted instrumentation techniques can help to describe how anatomical structures move relative to one another in environments the human body did not normally experience in it’s evolution.

Grant Funding Source: National Highway Safety Administration
Characterization of putative mutagenesis cassettes in Sinorhizobium meliloti (927.1)
Authors: Genardi Samantha , DeLateur Nicholas , Kramer Caitlin , Leifer Becky , Brewer Tess , Jones Kathryn , Beuning Penny
Published: June 2021

The goal of this research is to biochemically characterize chromosomal and plasmid‐encoded members of Y‐family DNA polymerases and putative mutagenesis cassettes from the nitrogen‐fixing plant symbiotic bacterium Sinorhizobium meliloti. Y‐family translesion polymerases are recruited to sites of DNA damage to replicate damaged DNA that may cause disruption to the replication fork. ImuC is a C family DNA polymerase with a putative function in mutagenic DNA replication; while ImuA and ImuB appear to be important for ImuC function, their roles have yet to be elucidated. S. meliloti harbors the Y family DNA polymerase dinB and the imuABC mutagenesis cassette encoded on both the chromosome and one of the symbiotic plasmids. All of the genes of the putative mutagenesis cassettes have been subcloned into two different plasmid vectors for in vivo and in vitro experiments. We are currently optimizing conditions for protein expression in E. coli. We are also carrying out assays to determine the ability of these gene products to confer on E. coli survival to a variety of DNA damaging agents and damage‐induced mutagenesis. Supported by NSF and ACS
One institution's approach: how the University of Texas at Austin merges research and teaching through the Freshman Research Initiative (93.1)
Authors: Simmons Sarah
Published: June 2021

The integration of research with education both engages scientists and improves courses, and is critical to improving American competitiveness in science. However, comprehensive curriculum reform must overcome organizational barriers and extreme inertia. Therefore, high‐impact models for improving curriculum and pedagogy must: 1) work within current educational structures and efforts, and 2) be flexible and adaptive enough to be implemented within different disciplines. The Freshman Research Initiative (FRI) at UT‐Austin is a faculty‐initiated reinvention of our undergraduate research paradigm recruiting over 800 freshmen each year into an integrated 3‐semester research‐based course sequence that aligns our research and teaching missions, increases the number and diversity of students engaged in research and significantly impacts student success and engagement in science. The FRI model is scalable, exportable, discipline‐independent and includes all aspects of research. One of the goals of FRI is to extend the opportunities for research experiences to students who have traditionally not chosen or not had access to such experiences, and by providing transformative educational experiences at scale to a large, diverse student body, we are contributing to a diverse pipeline of future scientists. Our program, its goals, impact data and potential for future expansion will be presented.
Cranberry (Vaccinium macrocarpon) extract inhibits matrix metalloproteinase expression in aortic smooth muscle cells by affecting key cell signaling pathways (934.1)
Authors: Hurta Robert , Thomas Amanda , Kim Joseph , Patel Kunal , Neto Catherine
Published: June 2021

A whole cranberry (Vaccinium macrocarpon) extract was shown to inhibit matrix metalloproteinase (MMP‐2 /MMP‐9) activity (assessed by zymography)in A7R5 aortic smooth muscle cells in vitro. Cranberry extract [CE] decreased cellular viability ~10% post 6 hours of treatment. Treatment of A7R5 cells with 100 ug/mL cranberry extract for 6 hours resulted in increased expression of TIMP‐1 & TIMP‐2 protein levels [inhibitors of MMPs] and decreased protein expression levels of EMMPRIN [an activator of MMPs]. In response to CE treatment, increased expression of JNK‐1, JNK‐2, pJNK‐1, pJNK‐2 & p38 protein levels occurred. No change in ERK‐1, ERK‐2 and pERK‐1 protein levels (assessed by Western blot analyses) occurred in response to cranberry treatment for 6 hours. However, a decrease in p‐p38 and in p‐ERK‐2 protein expression levels was noted in response to CE treatment.Treatment of A7R5 cells with CE (for 6 hours) also resulted in no apparent change in either AKT, pAKT, P‐I‐3 kinase p85 or p110 protein level expression. Treatment of A7R5 cells with CE (100 ug/mL) for 6 hours resulted in inhibition of FAK & p‐FAK protein expression levels . These results suggest that a whole cranberry extract has the ability to inhibit the expression of MMP‐2/‐9 activity in A7R5 aortic smooth muscle cells and suggests that this occurs via alterations in the expression of key protein modulators of MMPs’ expression and also involves changes in signal transduction pathways. (Canadian Institutes of Health Research [CIHR], P.E.I. Health Research Program, The Cranberry Institute ([Wisconsin Board ] funded).
Structures, dynamics, and interactions with UBC9 of the RING and B1‐box domains of promyelocytic leukemia protein (938.1)
Authors: Huang Tai‐huang , Huang Shu‐Yu , Naik Mandar
Published: June 2021

PML protein is mainly present inside the nucleus in the form of PML nuclear bodies (PML‐NB) and serves as anchoring point for various sumoylated transcription factors and regulators. It mediates activities, such as transcriptional regulation, antiviral defense, DNA replication, DNA repair, telomere lengthening, chromatin organization, cell cycle control, senescence, apoptosis, and tumor suppression. PML‐NB is associated with acute promyelocytic leukemia (APL). All known isoforms of PML have conserved N‐terminal RBCC region comprised of a RING finger domain followed by two B‐boxes and coiled‐coil region. The RING domain has been shown to possess SUMO E3 ligase activity and the B1‐box can be poly‐sumoylated. Both the RING domain and the B1‐box are zinc finger proteins. Surprisingly, only the structure of the RING domain was determined in 1995 and no information on the structure of other part of the molecule is known. Here we will present the solution NMR structures and dynamics of the RING domain and the B1 box, as well as their interactions with Ubc9, SUMO and SUMO substrates.

Grant Funding Source: Supported by a Grants NSC 101‐2311‐B‐001‐025 from The National Science Council
Organization of protein factors on the carboxy terminal domain of RNA polymerase II in yeast (941.1)
Authors: Mosaheb Mohammad , Fuchs Stephen
Published: June 2021

Organization of Protein Factors on the Carboxy Terminal Domain of RNA Polymerase II in Yeast Mohammad M. Mosaheb and Stephen M. Fuchs: Tufts University, Medford, MA, Department of Biology.

The Carboxy Terminal Domain (CTD) of RNA Polymerase II (RNAPII) in yeast is made up of 26 heptad repeats with the sequence YSPTSPS. The heptad repeats have different phosphorylation patterns at their 3 serines at different stages of transcription which mediate the recruitment of specific protein factors required for transcription. Our goal is to find out how the protein factors organize themselves on the 26 repeats ‐ Do the protein factors have to bind to a specific range of repeats? Or can they bind anywhere on the CTD and still be functional. For this work, we divided the CTD into 3 segments of 8 heptad repeats and subsequently created variants in which the serines in one of three segments were replaced with alanine. Measuring yeast growth on plates we have demonstrated that repeats likely have non‐redundant function. We are currently isolating RNAPII complexes to biochemically identify difference in protein factor abundance in our three mutants. We are also creating 3 new mutants heptad repeats each one with only one of the 3 serines mutated, these new mutants will be helpful to decipher which one of the 3 serines is more important in recruiting our protein factor of interest.
The effects of histone glutathionylation on chromatin structure (942.5)
Authors: Blackshire Bradley
Published: June 2021

The Effects of Histone Glutathionylation on Chromatin Structure DNA transcription is dependent on interactions between DNA and associated histone proteins. Modifications to histones have the potential to influence DNA transcription by promoting euchromatin structure, such as in the acetylation of histones, or promoting heterochromatin structure, such as in the methylation of histones. Recent work has increased our understanding of the consequences of histone modifications. In particular, a recent study demonstrated that cells exposed to nitrosative stress show an increase in histone glutathionylation, a redox sensitive modification. The current project focuses on the ramifications of glutathionylation of histones on chromatin structure and its potential to regulate gene transcription. We show that glutathionylation promotes heterochromatin structure, which may lead to a decrease in gene transcription. These results have broader significance as chromatin remodeling may play a role in aging and neurodegenerative diseases.

Grant Funding Source: STEM Title V