bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of intragastric infusion of copper sulfate on the responses of parabrachial nucleus neurons to whole‐body rotations in decerebrate felines
Authors: Suzuki Takeshi , Sugiyama Yoichiro , Yates Bill J
Published: June 2021

The parabrachial nucleus (PBN) plays a key role in relaying gastrointestinal (GI) signals to neurons in the limbic system and hypothalamus, and thus is likely involved in producing nausea. Some PBN neurons also receive vestibular inputs, and may participate in generating motion sickness‐related nausea. However, no previous studies have considered the interactive effects of GI inputs on PBN responses to whole‐body rotations. In the present study, we compared the responses of PBN neurons to rotations in vertical planes before and after the intragastric infusion of the emetic drug copper sulfate. The firing rate of a variety of PBN neurons, including those with respiratory‐related activity and those that received GI and baroreceptor inputs, was modulated by rotations in vertical planes. More PBN neurons were activated better by tilts in the sagittal (pitch) plane than by rotations in the transverse (roll) plane, and the responses of most of the cells were similar to those of otolith organs. For neurons with GI inputs, it was common for responses to vestibular stimulation to be affected by copper sulfate infusion: responses in 3/11 cells disappeared, whereas responses in 4/7 cells became evident. It was rare for copper sulfate infusion to affect the responses to vestibular stimulation of respiratory or cardiovascular units. These data show that emetic inputs alter the processing of motion signals by PBN neurons.
Increased Group I Metabotropic Glutamate Receptor Activity Contributes to Hyperactivity of Presympathetic Paraventricular Neurons in Hypertension
Authors: Li De-Pei , Pan Hui-Lin
Published: June 2021

Hypothalamic group I metabotropic glutamate receptors (mGluRs) importantly maintain elevated sympathetic vasomotor tone in hypertension. In this study, we determined the cellular mechanism underlying increased group I mGluRs in the control of excitability of paraventricular (PVN) presympathetic neurons in spontaneously hypertensive rats (SHR). The spinally projecting PVN neurons were retrogradely labeled by fluorescent microspheres injected into the intermediolateral cell column in the rat thoracic spinal cord. Group I mGluR selective agonist S‐DHPG caused a comparable dose‐dependent increased in firing activity in both Wistar‐Kyoto (WKY) and SHR. S‐DHPG also decreased transient outward K+ currents (IA) in the PVN neurons. Blocking IA with 4‐ aminopyridine markedly attenuated S‐DHPG‐induced increase in firing activity in WKY and SHR. IA currents were significantly lower in SHR than in WKY rats. Blocking mGluR5 receptor with selective antagonist MPEP decreased firing rate of PVN neurons in SHR, but not in WKY rats. Furthermore, MPEP produced an increase in IA currents in the PVN neurons in SHR. However, blocking mGluR1 receptor with selective antagonist LY367385 failed to alter the firing activity and IA currents in both WKY and SHR. These data suggest that increased group I mGluR activity tonically supports hyperactivity of PVN presympathetic neurons through suppression of IA currents in SHR.
The Effect of Hemorrhagic Shock and Resuscitation on Blood‐Brain Barrier Integrity
Authors: Hao Qi , Miyazawa Byron , Kutcher Matthew , Vilardi Ryan , Matthay Michael A. , Cohen Mitchell J.
Published: June 2021

Hemorrhagic shock (HS) is a primary cause of death after trauma. In many non‐brain injured patients, neurobehavioral changes have been observed after injury and HS suggesting distant effects on the brain from extracranial trauma. We here explore the effects of HS and resuscitation on blood‐brain barrier (BBB) integrity. Male C57BL/6J mice were subjected to HS at a MAP of 35mmHg for 60 min. Mice were then resuscitated with shed blood and lactated ringers solution. Mice were sacrificed at time after HS, and 2, 24, and 72 hours after resuscitation (HS+R). Mouse brain sections were immunohistochemically assayed for expression of the tight junction proteins ZO‐1 and Claudin‐5. In separate experiments, Evan's blue (EB) dye was injected via tail vein 1 hr before sacrifice and brain sections were examined for evidence of BBB permeability. ZO‐1 was greatly reduced in the mouse brain in HS alone, HS+R 2 hr and HS+R 24hr mice, with a marked restoration of signal in HS+R 72hr mice. Claudin‐5 expression remained unchanged. EB extravasation was observed in the mouse brain following HS, with the most notable extravasation seen in the hippocampus of HS+R 2hr mice. These results suggest that BBB permeability is increased following HS and resuscitation, likely mediated by decreased ZO‐1. Future studies will investigate the dynamic quantitative changes of BBB permeability and its mechanisms. Support: DOD W81XWH‐10‐1‐0509, NIH 5GM085689.
Differential Involvement of Tight Junction Associated Proteins in Regulating Blood‐Brain Barrier Endothelial Cell Permeability
Authors: Tharakan Binu , Sawant Devendra , Hunter Felicia A , Childs Ed W
Published: June 2021

Microvascular hyperpermeability of the blood‐brain barrier (BBB) leads to brain edema and neuroinflammation. The tight junction associated proteins of the BBB endothelial cells such as claudin‐5, occludin and zonula occludens‐1 (ZO‐1) are key molecules that regulate barrier integrity. Our objective was to determine if knockdown of various tight junction‐associated as well as adherens junction proteins in brain microvascular endothelial cells would lead to selective and differential barrier properties. Rat brain microvascular endothelial cell (RBMEC) monolayers grown on Transwell plates or on chamber slides were transfected with siRNAs for the tight junction associated proteins, claudin‐5, occludin and ZO‐1 and the adherens junction proteins β‐catenin and VE‐cadherin. FITC‐dextran flux across the monolayer was used as an indicator of the changes in permeability. The integrity of the monolayers were determined by using immunofluorescence localization of the above mentioned proteins followed by confocal microscopy. ZO‐1 siRNA trannsfected monolayers but not claudin‐ 5 or occludin siRNA transfected monolayers showed significant hyperpermeability (p<0.05). β‐Catenin or VE‐cadherin siRNA trasfected monolayers showed no significant change in permeability. Tight junction associated proteins of the BBB show differential involvement in regulating microvascular hyperpermeability. ZO‐1 plays a predominant role in maintaining the BBB integrity and permeability compared to the other tight junction associated and adherens junction proteins.
EETs pre‐treatment improves mitochondrial dynamics against Aβ‐induced damage in cultured hippocampal astrocytes
Authors: Sarkar Pallabi , Zaja Ivan , Bienengraeber Martin , Canfield Scott , Harder David R
Published: June 2021

The neurodegeneration seen in Alzheimer's disease (AD) is largely attributed to the over‐production and accumulation of the protein Amyloid beta (Aβ), which becomes toxic to the cells and induces dysfunction of cellular organelles to inflict the damage. Recent publications have demonstrated that the epoxide derivatives of arachidonic acid, epoxyeicosatrienoic acids (EETs; 14,15‐, 11,12‐, 8,9‐ and 5,6‐EET), are cytoprotective in the brain and promote neuronal growth. The involvement of EETs in AD has not been studied yet. Our previous data suggests that Aβ affects EETs production (Sarkar et al, Neuroscience, 2011, p241–249). In the current study, we evaluated the cytoprotective potential of exogenous EETs against Aβ damage in cultured astrocytes. Pre‐incubation with 14,15‐ and 11,12‐EET protected the cells against Aβ‐induced mitochondrial fragmentation and membrane depolarization. Moreover, EETs reversed the Aβ‐mediated uncoupling of electron transport chain from ATP synthesis and reduced cellular ROS production. Our data provides the first evidence of the involvement of EETs in the downstream pathway of Aβ toxicity and manipulating this signaling cascade may improve AD pathology. This study was supported by grants awarded to David R. Harder by the National Institute of Health (RO1 HL033833, RO1 HL092105) and also by the VA Research Career Scientist Award.
Mitochondrial fission blocker protects brain endothelial cells but not neurons following oxygen‐glucose deprivation (OGD)
Authors: Wappler Edina A , Katz Paige S , Katakam Prasad V.G. , Busija David W
Published: June 2021

The aim of our study was to investigate the effect of mitochondrial fission blocker Mdivi‐1 on cell survival following OGD in neurons and cerebral vascular endothelial cells. Primary rat cortical neurons were isolated from E18 Sprague Dawley fetuses, while primary rat brain endothelial cells (RBEC) were isolated from 2 week old pups. We also used a human brain endothelial cell (HBEC) line in this study. In the confluent cultures, cell viability was measured following 1, 2, and 3 hour of OGD in HBEC, RBEC, and neuronal cells, respectively, which was followed by 24 hour reoxygenation in each case. Each group had a normoxic control with the same treatment as the OGD plates. Cells were exposed to Mdivi‐1 (2.5 μM‐ 250 μM), an inhibitor of the mitochondrial fission protein, dynamin‐related peptide 1 (Drp1), during the OGD. Mdivi‐1 treatment improved cell viability following OGD by 15–45% in HBEC, and 15% in RBEC compared to untreated cells exposed to OGD. However, Mdivi‐1 failed to change the cell viability in the neurons exposed to OGD. In conclusion, the mitochondrial fission blocker Mdivi‐1, is protective against OGD in endothelial cells from both rat and human, but not in rat neurons.
Decreased Pancreatic Insulin Secretion is associated with increased AT1 Activation during Glucose Supplementation in a Model of Metabolic Syndrome
Authors: Rodriguez Ruben , Viscarra Jose A. , Minas Jacqueline N. , Lee Andrew , Nakano Daisuke , Nishiyama Akira , Ortiz Rudy M.
Published: June 2021

Blockade of the renin‐angiotensin‐system improves glucose intolerance, insulin resistance and beta cell function. The contributions of angiotensin receptor activation and glucose supplementation to impaired insulin secretion in the pathogenesis of metabolic syndrome are, however, not well defined. After 6 weeks of glucose supplementation and ARB treatment, an oral glucose tolerance test (oGTT) was performed to assess insulin resistance in 5 groups of rats: 1) untreated, lean LETO, 2) untreated, obese OLETF, 3) OLETF + ARB (olmesartan; 10 mg/kg/d; OLETF ARB), 4) OLETF + 5% glucose water (OLETF HG), and 5) OLETF + HG + ARB (OLETF HG/ARB). ARB treatment reduced the oGTT response 19% and increased pancreatic insulin secretion 64% and 113% in OLETF ARB and OLETF HG/ARB, respectively. ARB treatment in OLETF ARB and OLETF HG/ARB did not have an effect on insulin signaling proteins in skeletal muscle; however, it reduced pancreatic AT1 protein expression 20% and 27%, increased pancreatic GLP‐1r protein expression 41% and 88%, respectively, and increased fasting plasma GLP‐1 approximately 2.5‐fold in OLETF ARB. The results suggest that improvement of glucose intolerance is independent of an improvement in muscle insulin signaling, but rather by improved glucose‐stimulated insulin secretion associated with decreased pancreatic AT1 activation, and increased GLP‐1 signaling. (NIH NCMHD 9T37MD001480)
SIK3 is a new regulator of lipid homeostasis in the mouse liver
Authors: Ito Yumi , Uebi Tatsuya , Kumagai Ayako , Sanosaka Masato , Takemori Hiroshi
Published: June 2021

A life free from obesity is very appealing in today's modern society. Here, we report a new molecule that links cholesterol‐bile acid homeostasis with energy storage capacity. Salt‐inducible kinase 3 (SIK3), an AMP‐activated protein kinase‐related kinase, is induced in the murine liver after consumption of a diet rich in fat, sucrose, and cholesterol. Sik3−/− mice have a malnourished phenotype (i.e., lipodystrophy, hypolipidemia, and hypoglycemia) accompanied by cholestasis and cholelithiasis. These biliary disorders are correlated with the dysregulation of gene expression programs that respond to nutritional stresses and are probably regulated by nuclear receptors. Retinoid X receptor (RXR) plays a role in cholesterol and bile acid homeostasis, while ALDH1a, which produces the RXR ligand 9‐cis‐retinoic acid, is expressed at low levels in Sik3‐KO mice. The disorders of lipid metabolism in Sik3−/− mice are ameliorated by the treatment with 9‐cis‐retinoic acid. Moreover, the administration of adenoviruses expressing human SIK3 into Sik3−/− mice restores gene expression profiles responding to nutritional stresses, including the Aldh1a gene. In conclusion, SIK3 is a novel energy regulator that increases nutritional storage by facilitating the clearance of cholesterol and bile acids while leading to obesity.
High Dietary Glucose Intake Increases Hepatic Triglyceride Content and Oxidative Stress: Contributions of Angiotensin Receptor
Authors: Montez Priscilla , Max Thorwald A. , Rodriguez Ruben , Viscarra Jose , Vázquez-Medina José Pablo , Nakano Daisuke , Nishiyama Akira , Ortiz Rudy M
Published: June 2021

Excessive glucose intake exacerbates the deposition of adipose and plasma lipid levels and can subsequently increase organ‐specific oxidative damage during insulin resistance. To test our hypothesize that angiotensin receptor contributes to increased hepatic triglyceride content and oxidative stress following increased glucose intake in a model of metabolic syndrome, we studied 5 groups of rats: 1) LETO (lean control strain), 2) OLETF (15 wk), 3) OLETF + ARB (10 mg olmesartan mg/kg/d), 4) OLETF + high glucose (HG; 5% in water) and 5) OLETF + ARB + HG. HG had increased plasma triglycerides (TG) (35%), plasma glycerol (87%), free fatty acids (FFA) (28%) and body mass (BM) (37%), indicative of dislipidemia, as well as increased (74%) hepatic lipid peroxidation (4‐HNE). ARB treatment in HG reduced plasma TG (15%), plasma glycerol (23%), FFA (14%), BM (12%), and hepatic TG content (42%). ARB treatment in HG also decreased hepatic nitrotyrosine and increased UCP2 protein expression (59%), aconitase (40%) and antioxidant enzyme activities 50–120% suggesting that AT1 activation contributes to protein oxidation, lipid metabolism, fat deposition and lipid‐associated hepatic oxidative stress. Thus, besides the antihypertensive effects of ARB treatment, ARBs can also improve the condition of metabolic syndrome by improving the lipid profile and reducing adiposity and oxidative stress. Funded by NIH NCMHD 9T37MD001480 & P20MD005049.
Macrophage differentiation and functional polarization: role of thyroid hormones
Authors: Cervia Davide , Buldorini Marcella , Perrotta Cristiana , Clementi Emilio
Published: June 2021

Macrophages can be divided on basis of their function: classically activated macrophages (often referred to as M1) and alternatively activated macrophages (typically named M2). There is evidence that different endocrine stimuli influence macrophages differentiation with a mosaic of different and overlapping characteristics. In this study, we verified for the first time the role of thyroid hormone (TH) system on macrophage differentiation/polarization through in vitro and in vivo approaches. Monocites were isolated from femur bone marrow derived cells and primary cultures of differentiated macrophages (MΦ) were obtained. MΦ were also polarized to generate M1 and M2 subtypes. In these cells, we demonstrated different levels of TH receptors (TRalfa1, TRbeta1, and TRbeta2). Cells were then treated with TH during MΦ differentiation, as well as during polarization to M1 or M2, and the hormone‐induced effects were determined. For instance, a set of specific cell‐surface molecules and cytokine/receptor genes were measured. In addition, we determined TH effects on cell proliferation, cell viability, phagocytosis, nitric oxide synthase and arginase protein expression, cytokine release, and cell migration. In vivo experiments were also conducted in euthyroid and hypothyroid mice. In particular, we analysed the animal survival rate and the presence of cell‐surface molecules in peritoneal macrophages of mice both in control conditions and during systemic inflammation. Taken toghether, our data suggest that TH has a role in modulating specific properties of macrophage populations and the functional balance M1/M2.
Glucose and lipid metabolism are modified by short‐term alterations to milking frequency in grazing dairy cows
Authors: Grala Talia M , Phyn Claire V C , Kay Jane K , Rius Agustin G. , Littlejohn Mathew D. , Snell Russell G , Roche John R
Published: June 2021

Short‐term changes to milking frequency (once daily, 1X; or thrice daily, 3X) alter metabolic balance in dairy cows. There are contradictory results regarding effects when cows are switched to normal milking (twice daily; 2X). Cows were allocated to 3 or 6 weeks of 1X or 3X milking post‐calving, switched to 2X milking for the rest of lactation, and compared with cows milked 2X for the whole lactation. Serial liver and adipose biopsies were collected in the first 9 weeks (n=12 cows/trt) and gene expression measured by RT‐qPCR. Data suggests that cows milked 1X had reduced gluconeogenesis and β‐oxidation in liver tissue but increased β‐oxidation and lipolysis in adipose tissue during treatment, compared with cows milked 2X. Cows milked 3X for 6 weeks exhibited reduced lipogenesis and lipolysis in adipose tissue during and post‐treatment. Results indicate that alterations to milking frequency affect carbohydrate and lipid metabolism during, and after treatment. These results increase understanding of the regulation underlying responses to altered milking frequency, and may have implications for farm management. This research was funded through DairyNZ Inc. and the Ministry of Science and Innovation, NZ
CREAL: A language for describing biological systems from a macro to a molecular scale
Authors: Garan Steven A. , Sarcon Aida , Chen Alexander , Lu Erick , Gurm Hashroop K. , Gunther Sam , Zheng Ben C. , Zhu Julian , Banerjee Nabhojit , Govinthasamy Anensshiya , Neumann Rebecca , Freitag Warren , Brooks George A.
Published: June 2021

A language that can describe biological systems from a macro to a molecular scale can play an important role in better understanding the mechanisms that drive the aging process and other biological processes. The Center for Research and Education in Aging Language (CREAL), was developed to allow biologists and Artificial Intelligence (AI) systems to augment a Knowledge Base (KB) that can be used as a data source by a massively paralleled simulation engine to describe complex systems. CREAL uses Java Script Object Notation (JSON) as its template to create nodes of information that can interact with other nodes within the simulation. The language can describe systems of transportation and location as well as properties, actions, and conditions of biological components and processes. A simulation using a KB with CREAL nodes can reveal many relationships between processes when using Dijkstraˈs shortest path algorithm, and with the aid of tools such as Hidden Markov Models (HMM) could suggest possible linkages where currently none have been discovered, and could reveal new insights into phenomenon such as the aging process.
An Exercise to Transfer Learning to Novel Situations: The Student Perspective
Authors: Salci Lauren E. , Naji Faysal N. , Hoit Graeme S.
Published: June 2021

The Tri‐Partite Problem‐Solving Exercise (TRIPSE) is an evaluation method that simulates the scientific process. Students presented with limited data are required to state hypotheses, propose experimental tests to explore them and assess their answers after given additional information. The exercise has been used in class sizes ranging from 15 to 200 (FASEB Journal. 2008; 22:767.1). A variation, the Legacy TRIPSE (FASEB Journal. 2010; 24: 633.1) was later developed to engage students, encourage them to transfer learning to novel situations and create a bank of problems as a ‘legacy’ for future classes. Students designed problems based on published data, and provided suitable answers (hypotheses and experimental tests). We report the experience of the Legacy TRIPSE from the students’ perspectives. On a score of ten, students rated the project highly (median, mode, range, n). It provided a valuable learning experience (8, 10, 10, 100) and one that was significantly superior to conventional exams (8, 10, 10, 100). It allowed students to transfer concepts from lectures to novel situations, and helped them read scientific papers more critically and understand the operations of modern scientific practice.
Anatomical and radiographic characterization of the lateral patellofemoral ligament of the knee
Authors: Marberry Kevin , Boehm Karl , Korpi Fredrick , Kondrashov Peter
Published: June 2021

The purpose of this investigation was to isolate and characterize the patellar and femoral insertions of the Lateral Patellofemoral Ligament (LPFL) and define the corresponding radiographic landmarks. Ten embalmed cadaveric knees were dissected in order to examine the lateral patellofemoral structures. The LPFL was isolated and the surrounding soft tissue was removed. Measurements of the ligament were obtained with digital calipers. The patellar and femoral insertions of the ligament were identified and characterized based on their positions relative to known anatomic landmarks. Each specimen was examined radiographically after a radiopaque marker was placed at the center of the femoral insertion of each of the ligaments. The average center of the femoral insertion of the LPFL was calculated in reference to radiographic landmarks, including the Blumensaat's line and the posterior surface of the femoral cortex. The patellar insertion of the LPFL was found to consistently encompass the entire lateral margin of the patella. The data was analyzed using regression analysis. This allowed us to formulate an equation to determine the center of the femoral attachment of the LPFL based on measurements taken from a lateral and AP radiographs of each femur. Research support was provided by the Department of Anatomy at A.T. Still University.
Two nerve morphometry methods performed through the same computer program: are they comparable?
Authors: da Costa Bilego Neto Antônio Paulo , Silveira Fernando Braga Cassiano , Sanada Luciana Sayuri , Fazan Valéria Paula Sassoli
Published: June 2021

Morphometry has been widely used in studies designed to establish normal patterns of biological structures or to compare changes resulting from pathological processes. It can be performed manually or through semi‐automatic or automatic methods. However, it is difficult to compare results between studies that used different methods. We aimed to compare the results of two different methods of nerve morphometry, automatic and semi‐automatic, using the same computer software. Female Wistar rats (n = 5) with 90 days of age had their right and left sural nerves prepared for light microscopy. Automatic and semi‐automatic morphometry was performed using the program KS‐400 and took into consideration: area and lesser diameter of myelinated fibers and their axons, the myelin sheath area and the ratio between the lesser diameter of the axon and its respective myelinated fiber (G‐ratio). The nerves are longitudinally and laterally symmetric with both morphometric methods. A significant overestimation of the myelinated fibers size and a significant underestimation of the myelinated axons size were observed with the automatic method. Our results indicate an important differences in the values of the myelinated fibers morphometry of one nerve, obtained with two different methodologies. Thus, the same methodology must be standardized and used throughout a single experiment.

Grant Funding Source : FAPESP, CNPq and FAEPA
Ultrastructural investigation of the aortic depressor nerve in Wistar and Wistar‐Kyoto rats
Authors: de Amorim Milena Menezes , da Silva Letícia Oliveira Neri , de Aguiar Mendes Vânia Alice , Salgado Helio Cesar , Fazan Valéria Paula Sassoli
Published: June 2021

Ultrastructural study of the aortic depressor nerve (ADN) [1] showed differences on the morphology and morphometry between normotensive Wistar‐Kyoto (WKY) and spontaneously hypertensive rats (SHR), particularly in the number of unmyelinated fibers. The aim of the present study was to investigate if there are ultrastructural differences on the AND of two different normotensive rats strains. Twelve Wistar rats (WR) and 13 WKY had their left ADN fixed in 2,5% glutaraldehyde. Thin cross sections of proximal and distal segments of the ADN were studied by transmission electron microscopy and a morphometric study of the unmyelinated fibers was performed. Comparison between proximal and distal segments of the two strains revealed a longitudinal symmetry. Comparison between strains revealed that, despite the fact that WR are heavier (body weight = 339 ± 17g for WR and 277 ± 54 for WKY) than WKY, ADN unmyelinated fiber number and size is significantly larger in WKY. Unmyelinated fiber distribution is unimodal in both segments for both strains but WKY distribution was skewed to the right with a high percentage of fibers larger than 1.5 μm. Average values of unmyelinated fibers morphometric parameters are higher in WKY due to a larger number of fibers bigger than 1.5 μm.

Grant Funding Source: FAPESP, CNPQ, CAPES and FAEPA
Morphological characterization of the liver in a murine non‐alcoholic fatty liver disease model
Authors: Itagaki Hiroko , Kitahara Shuji , Morikawa Shunichi , Ezaki Taichi
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) is increasing and may progress to hepatocellular carcinomas. We sought to clarify the morphological and functional characteristics of the hepatic changes in a murine NAFLD model. Nine‐week(wk)‐old male C57BL/6 mice were fed an MCD (methionine‐chorine deficient) diet up to 30 wk (group M). Recovery model mice (group R) were prepared by feeding normal control diet for two weeks after the termination of MCD diet feeding for 8, 16, 30 wk. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were measured and liver cryosections were made. A multiple‐immunostaining technique for various markers was employed for the 3‐dimentional imaging by the confocal laser‐scanning microscopy. Serum AST was elevated from 2 wk (Wk 2) to Wk 16, but decreased at Wk 30 in group M. Oil red O staining also indicated that the group M caused fat accumulation in hepatocytes from Wk 2 to Wk 16. Fibrosis started from Wk 10 and progressed thereafter in group M. In group Rs, serum AST decreased and the fat deposition and fibrosis improved. The immunohistochemical data revealed swollen CD68 positive cells appeared and increased after Wk 16. In semi‐thin sections, these cells contained various‐shaped contents including dead cell fragments, and they remained in group R. The significance of these CD68 positive cells in the pathogenesis of the hepatic changes caused by the MCD diet is discussed.
A new regulatory pathway of mRNA export by an F‐box protein, Mdm30
Authors: Durairaj Geetha , Bhaumik Sukesh R.
Published: June 2021

Regulatory role of ubiquitylation has been implicated in various steps of gene expression and mRNA export is not an exception. Specificity of this modification is conferred by E3 ligases whose function is dictated by specific domains they possess. Role of HECT domain containing E3 ligases like Tom1 and Rsp5 have previously been implicated in regulation of mRNA export. However, pertaining to diversity of E3 ligases, the roles of other ligases in this aspect of gene expression is unknown. Interestingly, our recent work established the role of an F‐box protein, Mdm30, in stimulation of mRNA export. F‐box proteins are bonafide members of RING domain containing SCF (Skp1‐Cullin‐F box) ligase complex. To elucidate the mechanism‐of‐action of Mdm30, we carried out a series of experiments. We find that Mdm30 ubiquitylates the Sub2 component of Transcription/Export complex (TREX) in SCF dependent manner. Ubiquitylated‐Sub2 is subsequently degraded by 26S proteasome complex. Such targeted degradation of Sub2 enhances the recruitment of mRNA export adaptor, Yra1, onto active gene to promote mRNA export. Collectively, these results provide a new regulatory pathway of mRNA export, and will be presented in this conference.

Funding: Excellence in Academic Medicine (EAM) from the School of Medicine, Southern Illinois University.
CAP INDEPENDENT TRANSLATION CONTROLS IN BARLEY YELLOW DWARF VIRUS (BYDV)
Authors: Banerjee Bidisha , Sharma Sohani Das , Goss Dixie J.
Published: June 2021

BYDV has economic importance because it infects a broad range of cereal crops and can severely limit food grain production. Little is currently known about the mechanism of cap‐independent translation (CIT), an alternate means employed by many plant viruses e.g. BYDV to circumvent host defense mechanisms. Protein‐RNA complexes formed during the translation processes are dynamic and binding/release kinetics influence the sequential assembly of the initiation complex.

The CIT mechanism was investigated by studying the role of eukaryotic initiation factors (eIF) eIF4F, eIF4B, eIF4A and PABP in the translation initiation complex formation using the biophysical techniques fluorescence anisotropy and stopped flow kinetics. BYDV translation element (BTE) located in 3’ UTR (Untranslated RNA) of mRNA and a non‐functional mutant BTEBF bound eIF4F with equal binding affinity (Kd). The presence of other eIFs had a negligible effect on the equilibrium binding of eIF4F to BTE/BTEBF RNA. Recently obtained data indicate that the BTE (first order rate constant kobs= 12.3±0.2 sec−1) bound eIF4F three times faster than the un‐translating BTEBF (kobs= 4.4+0.5 sec−1) suggesting that kinetics play a role in the translation of these RNAs.
Mastermind‐like 1 is ubiquitinated: functional consequences for notch signaling
Authors: White Brandon , Farshbaf Mozhgan , Lindberg Mikael J. , Behmner Stefanie , Wallberg Annika E
Published: June 2021

The Mastermind‐like (MAML) proteins are transcriptional coactivators for Notch signaling, an evolutionarily conserved pathway that plays several key roles in development and in human disease. The MAML family contains MAML1, MAML2, and MAML3. We have identified MAML1 to be ubiquitinated. Deletion analysis shows that ubiquitination occurs in the first 301 amino acids of MAML1. MAML1 half‐life was calculated to be ~3 hours which is similar to half‐life reported for Notch. Overexpression of p300 stimulates ubiquitination of MAML1, but is independent of the p300 acetyl‐transferase activity. Finally, overexpression of Notch with MAML1 decreases ubiquitination. These results suggest that MAML1 may be turned‐over to maintain low protein levels in the absence of Notch. Upon Notch cleavage and translocation to the nucleus, MAML1 becomes more stable to help form the ternary complex for transcriptional activation of target genes. This research was supported by an NSF‐RUI Grant#1052039 to JBW, and grants from the Swedish Research Council, the Swedish Cancer Society, and the Swedish Children's Cancer Foundation to A.E.W. M.J.L. was supported by a fellowship from the Swedish Children's Cancer Foundation and a grant from Alex and Eva Wallstrom foundation.
MOLECULAR BASIS OF RNA POLYMERASE II TRANSCRIPTION FIDELITY
Authors: Wang Dong
Published: June 2021

A long‐standing key question in the transcription field has been how Pol II achieves such a high transcriptional fidelity, with an error rate ranging between 10‐4 and 10‐5.

Maintaining transcriptional fidelity is vital to cells and organisms. Novel insights into this subject have recently emerged from a combination of structural, genetic and biochemical studies, suggesting that the high accuracy of Pol II transcription is achieved by specific nucleotide selection and by removal of misincorporated nucleotides (proofreading). Here, we use a combined approach including pre‐steady state transient kinetics, X‐ray crystallography, and molecular dynamics simulations to elucidate the chemical principles in controlling Pol II transcription fidelity.
Nonpolar nucleosides alter RNA Polymerase II NTP specificity by disrupting hydrogen bonding and base stacking
Authors: Kellinger Matthew William , Ulrich Sebastien , Kool Eric T , Wang Dong
Published: June 2021

RNA Polymerase II (RNAPII) catalyzes the synthesis of mRNA during transcription. RNAPII nucleotide discrimination is governed by the relative specificity constants (kcat/Km) for correct versus incorrect nucleotide substrates. We have investigated the mechanisms governing nucleotide specificity by substituting the DNA templates with nonpolar nucleosides containing C2 and C4 halogen substitutions of increasing size. These substitutions disrupt hydrogen bonding by removing charged groups that interact with the incoming correct ATP during traditional Watson‐Crick base pairing. Our findings reveal nonpolar analogs greatly decreases the specificity constant for correct ATP incorporation. This decrease in specificity also abolishes the polymerases ability to discriminate against incorrect UTP incorporation. We further determine the contribution of hydrogen bonding on next nucleotide incorporation. The results indicate that the absence of 3′ hydrogen bonding decreases mismatch recognition.
Srs2 functions needed to replicate CAG/CTG hairpins and prevent repeat instability
Authors: Nguyen Jennifer , Anand Ranjith , Richard Guy-Franck , Freudenreich Catherine
Published: June 2021

Trinucleotide repeats form secondary DNA structures that can lead to chromosomal instability, breakage, and fork stalling during replication. Srs2, a non‐replicative helicase, anti‐recombinase that can displace Rad51, and interacts with PCNA, is required for reversed fork formation, which facilitates replication through these structures and prevents trinuleotide repeat expansion. However, how exactly Srs2 contributes to fork reversal and repeat stability still remains elusive. To approach this problem, we tested Srs2 mutants deficient in specific domains, which allowed us to investigate the uncoupling of each domain and how each individually contributes to maintaining genome integrity. We tested these mutants with a PCR based stability assay and fragility assay. We show that Srs2 unwinding and Rad51 displacement activities are all required to prevent CAG/CTG repeat expansions, contractions, and chromosomal breakage. We also show that its PCNA interacting domain is critical in preventing chromosomal breakage but not required to prevent repeat instability or fork reversal. Together, these results suggest that there is an uncoupling of Srs2 domain requirement in genome maintenance. Analysis by 2D electrophoresis will further shed light on which activities contribute to fork reversal. This research was funded by National Institutes of Health grant GM063066 and Tufts Department of Biology
Investigating the role of sterics and hydrogen bonding in the preQ1 riboswitch binding site
Authors: Ryon Lauren W , Suydam Ian T
Published: June 2021

We have investigated the relationship between metabolite structure and binding affinity in the Bacillus subtilis preQ1 riboswitch. Chemical modifications were introduced at the 7‐aminomethyl position of preQ1 to explore the role of hydrogen bonding and sterics in the riboswitch binding site. Analogs were prepared via a Mannich reaction of 7‐deazaguanine, followed by amine exchange with primary and secondary amines. The binding affinity for each analog was determined by monitoring the folding induced change in fluorescence of a 2‐aminopurine modified RNA. Measured binding affinities were highly dependent on analog structure. Alkylation at the 7‐aminomethyl position led to decreases in binding affinity between 20 and 1,000 fold. These results confirm the high selectivity of the preQ1 riboswitch for its natural substrate. This work was supported by The Murdock Trust, The College of Science and Engineering of Seattle University and The Department of Bioengineering of The University of Washington.
Synergy Between Chromatin Remodeling Complexes and pre‐mRNA Splicing
Authors: Pradhan Suman Kalyan , Soule Erik , Johnson Tracy L
Published: June 2021

Pre‐messenger RNA splicing plays a critical role in gene expression. In spite of the considerable progress that has been made in understanding the mechanism of splicing, an important question that remains poorly understood is how changes in the chromatin template affect splicing. Observations of enriched nucleosome occupancy and histone modifications in the exonic regions of genes raise the possibility that specific histone marks may be associated with splicing signals. For example, proteins that bind to methylated histones facilitate the recruitment of snRNPs to nascent transcript and influence the efficiency of splicing. Recently, we have shown that GCN5‐dependent histone acetylation is critical for cotranscriptional spliceosome assembly and its rearrangements. To gain further mechanistic insights, we have explored the possibility of communication between the chromatin remodeling machinery that interact with acetylated histones and the splicing machinery. Genetic analyses have identified functional interactions between components of chromatin remodeling complexes and components of the U2 snRNP. Furthermore, in vivo interaction studies demonstrate physical interactions between the Swi/Snf complex and the U2 snRNP. Together, these studies suggest a synergistic relationship between the Swi/Snf complex and the splicing machinery in gene expression.

Funding: NIGMS (GM085474 and GM085764)
Cathepsin B cleaves and activates the alpha subunit of the epithelial sodium channel
Authors: Alli Abdel A , Song John Z , Al-Khalili Otor , Bao Hui-Fang , Ma He-Ping , Eaton Douglas C
Published: June 2021

Several proteases have been found to cleave ENaC subunits to produce the active form of the heterotrimeric channel. ENaC cleavage is thought to occur primarily in the trans‐Golgi network (TGN) by serine proteases. ENaC may undergo additional cleavage after it is inserted into the apical membrane or while it is being recycled in recycling endosomes. Therefore, we investigated if secreted proteases could cleave and activate ENaC subunits. We performed two‐dimensional gel electrophoresis after concentrating condition medium taken from the apical and basolateral sides of 2F3 cells that formed tight junctions and demonstrated a tight resistance. We identified a protein secreted onto the apical side of 2F3 cells by mass spectrometry, as the cysteine protease cathepsin B. We expressed and purified full length recombinant alpha, beta, and gamma subunits of ENaC as GST fusion proteins. We identified a putative site for cathepsin B within the alpha subunit of ENaC that is somewhat conserved between species. We demonstrate that active cathepsin B cleaves the alpha subunit, but not the beta or gamma subunits of ENaC in vitro. We use single channel patch clamp studies and short circuit current experiments to show ENaC activity decreases in response to a specific cathepsin B inhibitor after being directly applied onto the apical side of 2F3 cells. These results suggest a novel mechanism for the proteolytic regulation of ENaC.
The role of protein oxidation in the onset of the transthyretin amyloidoses
Authors: Zhao Lei , Reixach Natalia , Buxbaum Joel
Published: June 2021

The transthyretin (TTR) amyloidoses are a subset of protein misfolding diseases characterized by the age‐dependent deposition of amyloid fibrils, derived from either wild type or mutant forms of the plasma protein TTR. Although it is clear that age is a major risk factor for the development of the TTR and other systemic amyloidoses, there are few studies addressing the molecular mechanisms responsible for the apparent effect of aging.

There is a general increase in the concentrations of oxidation products of proteins, lipids and DNA associated with aging. We hypothesize that an increase in TTR oxidation with age predisposes to TTR aggregation in vivo. We have produced oxidized forms of wild‐type TTR similar to those reported to be present in human plasma. Chaotrope‐mediated denaturation/renaturation experiments showed that modified forms of TTR, including those with oxidized methionines and cysteines and those with carbonylated amino acids displayed reduced in vitro stability relative to native non‐oxidized TTR. The modified TTRs also had differential propensity for amyloid fibril formation with respect to native TTR. Sera from mice transgenic for a wild‐type human TTR gene show increased degrees of TTR carbonylation with aging. The greater proportion of circulating oxidized TTR seen in the older mice with its lower stability, may play a role in the development and onset of the senile wild type TTR associated amyloidosis. (This work is supported by 1R01AG032285 NIH/NIA)
Regulation of the Get3 ATPase Cycle
Authors: Rome Michael Evan , Rao Meera
Published: June 2021

The cytosolic ATPase Get3/TRC40 mediates targeting of tail‐anchored (TA) membrane proteins to the endoplasmic reticulum (ER). Get3 functions as a molecular chaperone by binding to transmembrane domains of newly synthesized TA proteins and delivering them to a membrane docking complex in the ER. Previous work have shown that ATP binding and hydrolysis can regulate Get3's function and are essential for the targeting of TA proteins. However, the precise mechanisms by which this is accomplished remain unclear. Using a combination of mechanistic enzymology and biophysical methods, we delineate Get3's ATPase cycle and show that it is required at multiple stages during TA protein targeting. Our data complements previous structural studies and contributes to our understanding of how Get3 harnesses the energy of ATP hydrolysis to spatially and temporally coordinate the delivery of TA proteins to the ER membrane.
Complementary rather than redundant CYP3A4 ubiquitination by UBC7/gp78 and UbcH5a/CHIP E2/E3 systems
Authors: Wang YongQiang , Correia Maria Almira
Published: June 2021

We have shown that UBC7/gp78 and Hsp70/UbcH5a/CHIP E2‐E3 ligases both catalyze CYP3A ubiquitination in vitro and in vivo and target it to 26S proteasomal degradation. To determine whether such E3 function was complementary or redundant, we employed in vitro CYP3A4 ubiquitination systems that contained all the required components for each E3‐ligase either simultaneously in the 90‐min incubation (System 1), or a corresponding system that excluded either of the two specific cognate E2s (UBC7 or UbcH5a) just during the first 30 min of incubation (System 2 or 3). The E2 was then supplemented and the reaction carried out to its 90‐min completion. Individual E2/E3 incubations were also run in parallel (Systems 4 and 5). We found that CYP3A4 ubiquitination was >10‐fold enhanced in System 1 containing the complete system from the start relative to either individual System 4 or 5. However, exclusion of UbcH5a (System 3) in the first 30 min greatly impaired the overall extent of CYP3A4 ubiquitination due to appendage of fewer and/or shorter ubiquitin chains, relative to that observed on corresponding exclusion of UBC7 (System 2). However the CYP3A4 levels modified in either System 2 or 3 were comparable. These findings reveal that each E3 ligase is complementary rather than redundant in CYP3A4 ubiquitination: CHIP is required for initial ubiquitination and gp78 functions as an E4 in chain elongation. Supported by NIH grant GM44037.
Developing polyketide‐based anti‐microbial therapeutics using synthetic enzymology
Authors: Cheung Vivian Wing Ngar , Go Maybelle Darlene Kho , Robinson Robert , Wongsantichon Jantana , Yew Wen Shan
Published: June 2021

Enterococcus faecalis is an example of a bacterium that has gained multi‐drug resistance and is a major cause of nosocomial infections. In this project, we are targeting the enzymes present in the shikimate pathway within E. faecalis, as this pathway is absent in humans and crucial to the microbe, making the pathway an attractive target for drug design. A preliminary screen using a limited compounds library was performed on two enzymes present within the shikimate pathway and several potential inhibitors bearing the polyketide scaffold were identified. Polyketides are a large class of natural biomolecules biosynthesized from acyl‐CoA precursors using polyketide synthases (PKSs) that includes clinically important antibiotics such as tetracycline. Our project focuses on an uncharacterized Type III PKS, a putative chalcone synthase, from Oryza sativa (OsCHS). In order to produce a chemically diverse library of polyketides, a precursor‐directed biosynthesis approach was employed whereby different acyl‐CoA esters were tested as possible substrates for OsCHS and the enzyme was found to be promiscuous. The results suggested that the synthesis of novel compounds using synthetic enzymology was feasible and these polyketides could be used to screen for inhibitory activity against E. faecalis shikimate pathway enzymes to identify lead compounds for future antibiotic drug development.
Characterization of a Chitinase Activity in the Gut of the Yellow‐Bellied Slider Turtle Trachemys scripta
Authors: Kaiser Katie Ann , Tansey John T. , Bouchard Sarah S.
Published: June 2021

The Slider Turtle, Trachemys scripta, undergoes an ontogenetic dietary shift from a largely insect based diet to a plant based diet. Throughout the shift, the turtles must be able to access and utilize the maximum nutritional content available in both diets. Digestive enzymes such as endochitinase and exochitinase have been found throughout the gut that assist in nutrient breakdown. This work aimed to isolate and characterize the enzyme chitinase found to be optimally active at pH values 2.5 and 5.0 in T. scripta. The two activities suggest the presence of a cofactor, differing mechanisms, or two separate enzymes at work. Exochitinase activity was assayed using an N‐acetyl‐β‐D‐glucosaminide substrate esterified with fluorescent 4‐methylumbelliferone, which yielded a fluorescent product upon cleavage by the enzyme. Salt precipitation with 90% saturated (NH4)2SO4 and ion exchange chromatography were two techniques used to attempt to isolate activity at either pH 5.0 or pH 2.5. Enzyme activity at pH 5.0 was precipitated using the 90% (NH4)2SO4 solution but at pH 2.5 only half of the original enzymatic activity was recovered. This work was sponsored by the Student Research Fund of Otterbein University.
Structural insights into the mechanism of acetolactate decarboxylase
Authors: Marlow Victoria , Rea Dean , Wills Martin , Fülöp Vilmos
Published: June 2021

Acetolactate decarboxylase (ALDC) is a bacterial enzyme of the butanediol fermentation pathway that decarboxylates S‐acetolactate into R‐acetoin. The remarkable feature of this enzyme is that it also catalyses the decarboxylation of the opposite enantiomer R‐acetolactate to give the same product R‐acetoin. The enzyme is widely used commercially by the brewing industry. It shortens the maturation step by preventing the formation of diacetyl, which gives an off taste to beer. The structure of Bacillus brevis ALDC has been solved using X‐ray crystallography, in the presence of a series of chiral substrate analogues. In the resting state the active site zinc ion is coordinated to three conserved histidines and a glutamate. Upon binding of the substrate analogues the C‐terminal loop moves away losing the glutamate coordination bond. The structural information combined with experimental results from active site mutants allows us to present a mechanism for the stereospecific decarboxylation of acetolactate. The structural and biochemical data we have obtained will be used to develop further modifications and applications of the enzyme in future work.

The authors thank EPSRC for funding through the Molecular Organisation and Assembly in Cells (MOAC) Doctoral Training Centre.