bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modeling of the PTF1‐J and PTF1‐L Heterotrimeric Transcription Factor Complexes
Authors: Coats Ward 
Published: April 2020

Students in Biomedical Research Studies at Thomas Jefferson High School in Dallas, Texas participate in an ongoing research program in collaboration with the University of Texas Southwestern Medical Center to study the structure and function of a transcription factor complex. Ptf1a is a transcription factor that is crucial to the development of the embryonic pancreas. Its functional form is in a trimeric complex composed of a heterodimer of E12/E47 and Ptf1a (basic helix‐loop‐helix transcription factors), and either Rbpj or Rbpjl (Rbpj is also present in Notch signaling). The Rbpj form of the complex (PTF1‐J) is required for the early stage of pancreatic development. Subsequently, the Rbpjl‐form (PTF1‐L) is required for the formation of mature acinar cells of the exocrine pancreas. The Thomas Jefferson students using 3D printing technology has generated models for the PTF1‐J and PTF1‐L transcription factor complexes. The models provide the orientation of the Ptf1a/E12 heterodimer relative to the Rbpj and Rbpjl proteins and characterizes interactions formed between Ptf1a (tryptophan 298) and a hydrophobic pocket present in the beta‐trefoil domain of Rbpj or Rbpjl. Determination of the structures for the PTF1‐J and PTF1‐L complexes will help elucidate the structural determinants and the mechanism for the strong cooperative DNA binding of these complexes.Support or Funding InformationDallas Junior League: Grant for Innovative TeachingTexas Medical Association Award for Excellence in Science Teaching
Preliminary structural characterization of a methyltransferase involved in rhodoquinone biosynthesis
Authors: Vergunst Kathleen , Bergin Kathleen , Roger Andrew , Shepherd Jennifer , Langelaan David 
Published: April 2020

The primary process of energy production for most eukaryotes involves oxidative respiration via the electron transport chain (ETC). In the ETC, electrons are moved by the electron carrier ubiquinone through membrane‐bound protein complexes that pump protons across the mitochondrial membrane, which creates a proton chemical gradient. This gradient is used to produce adenosine triphosphate, which is the primary source of energy in the cell. Oxygen is the final electron acceptor of the ETC for aerobic organisms, but for organisms that live in low‐oxygen environments, other strategies are required for the ETC to function. One strategy used by many species of bacteria and protists is to produce rhodoquinone, which is a derivative of ubiquinone. Rhodoquinone allows the ETC to function in the absence of oxygen by allowing it to use fumarate as the final electron acceptor. RQ is synthesized by the enzyme RquA, which likely uses ubiquinone as a substrate. Our goal is to structurally characterize RquA to better understand its mechanism of catalysis. We used E. coli to overexpress RquA derived from Pygsuia biforma andRhodospirillum rubrum with a variety of fusion tags and found that RquA expresses to a high level but is located in inclusion bodies. We have developed refolding procedures to isolate RquA and are now characterizing its structure as well as the substrates required for RQ production.Support or Funding InformationThis research is funded by the New Frontiers in Research Fund ‐ Exploration Stream
In vitro and in vivo evidence for the role of lipid rafts in Ca2+‐gating of the Transient Receptor Potential channels in sensory neurons
Authors: Szőke Éva , Horváth Ádám , Bíró-Sütő Tünde , Sághy Éva , Payrits Maja , Erostyák János , Makkai Géza , Szolcsányi János , Helyes Zsuzsanna 
Published: April 2020

Transient Receptor Potential (TRP) cation channels as TRP Vanilloid 1 and TRP Ankyrin 1 (TRPV1 and TRPA1) are nocisensors playing important role to trigger pain. These cation channels serve as thermosensors and several exogenous and endogenous chemical ligands activate them. Capsaicin and resiniferatoxin (RTX) activate TRPV1, allyl‐isothiocyanate (AITC) and formaldehyde activate TRPA1 receptors. Lipid rafts are defined as liquid ordered plasma membrane microdomains rich in cholesterol, sphingomyelin and gangliosides. Sphingomyelinase (SMase) decreases membrane sphingomyelin by hydrolization, myriocin inhibits the glycosphingolipid synthesis. Our aim was to examine their in vitro actions on TRP channels, and the potential analgesic effect of SMase and myriocin in in vivo mouse models.The effect of SMase and myriocin was analysed on isolated trigeminal (TG) neurones by measuring agonists‐induced Ca2+ transients with ratiometric technique, and on TRPV1‐ or TRPA1‐expressing CHO cells by measuring 45Ca‐uptake. Mechanonociceptive and thermonociceptive thresholds of the animals in RTX‐induced thermal, and mechanical hyperalgesia, and formaldehyde‐evoked hyperalgesia model were measured. The analgesic effect of SMase and myriocin was measured in capsaicin‐evoked acute nocifensive response test.It has been revealed that intracellular Ca2+ enhancement evoked by capsaicin (TRPV1), AITC and formaldehyde (TRPA1) was inhibited after SMase and myriocin incubation in TG neurons and receptor‐expressing cells. Treatment with SMase and myriocin significantly diminished the RTX‐induced thermal, and mechanical hyperalgesia, the formaldehyde‐evoked hyperalgesia and the capsaicin‐evoked nocifensive reaction.On the basis of all these findings it can be concluded that the lipid membrane environment modulates the function of TRPV1 and TRPA1 channels and lipid raft interfaces modulate the channel opening. Targeting this interaction might be a tool for drug developmental purposes.Support or Funding InformationJános Bolyai Fellowship, KTIA_NAP_ 20017‐1.2.1‐NKP‐2017‐00002, GINOP‐2.3.2‐15‐2016‐00050, EFOP‐3.6.2‐16‐2017‐00008, 20765/3/2018 FEKUTSTRAT
Craniofacial growth and development in a mouse model of Down syndrome
Authors: Singh Nandini , Reeves Roger H. , Richtsmeier Joan T. 
Published: April 2020

Down syndrome (DS) is a developmental disorder caused by trisomy of human chromosome 21. Ts65Dn, a widely used mouse model of DS, has facilitated the study of mechanisms underlying the genotypic and phenotypic anomalies associated with DS. Previous studies have shown the adult cranium and brain to be markedly smaller in size in Ts65Dn mice relative to their euploid littermates. To determine how postnatal developmental patterns contribute to cranial variation in trisomic mice, we quantitatively compared growth trajectories from birth (postnatal day 0, P0) to postnatal day 6 (P6) in Ts65Dn and euploid mice. We hypothesized that trisomy affects cranial growth rate in Ts65Dn mice, causing size and shape differences between Ts65Dn and euploid mice. Multivariate regression and principal components analyses of craniofacial shape and size show some subtle differences between the trisomic and euploid mice at P0, but by P6 the trisomic mice are considerably smaller than the euploids. Interestingly, Ts65Dn at P6 are closer in both size and shape to P0 euploid mice than to euploid mice at P6 or newborn Ts65Dn. This could occur through changes in growth rate of Ts65Dn mice, resulting in the trisomic P6 mice retaining features common to younger, typically developing mice. Further research focusing on prenatal ages will provide a better understanding of how differences in timing of developmental events, such as delays in migration and mitosis of cranial neural crest cells in mouse model of DS, contribute to trisomic craniofacial growth and development.Support or Funding InformationR01‐HD038384 (RHR)
Single cell RNA sequencing (scRNAseq) of severely atherosclerotic coronary artery from Ossabaw miniature swine with metabolic syndrome
Authors: Eggenberger Caleb , Badin Jill K. , Alloosh Mouhamad , Sturek Michael 
Published: April 2020

IntroductionCoronary atherogenesis involves multiple signaling pathways and underlying gene expression. The atherogenic plasma milieu (excess lipoproteins, glucose, etc.) injures the endothelium, which is the initiating event of coronary plaque development. Subsequently, smooth muscle cells proliferate and migrate from the wall into the neointima, and blood cells and macrophages infiltrate the vessel wall and contribute to the growing plaque. Our goal was to characterize the gene expression in the heterogeneous cell population in this dynamic coronary artery pathology.MethodsWe performed single cell RNA sequencing (scRNAseq) on cells dispersed from the proximal 45 mm of the left anterior descending coronary artery of healthy pigs compared to separate plaque and wall cell dispersions from arteries of Ossabaw swine that had metabolic syndrome and were atherosclerotic for 23 months. Primary analysis scRNAseq data used t‐distributed stochastic neighbor embedding (T‐SNE) plots for an integrated analysis of the 3 cell preparations.ResultsMetabolic syndrome was confirmed by obesity, dyslipidemia, and hypertension. In vivo intravascular ultrasound (IVUS) of the entire artery revealed every stage of atherosclerosis from fatty streak to complex lesions having up to 70% occlusion with fibrofatty and calcified lesions. Cells (3,510) from healthy coronary artery expressed predominantly markers for endothelial (PECAM1), endocardial (CDH5, POSTN), and smooth muscle cells (ACTA2, TAGLN, CNN1, MYL9, CALD1, RGS5, MHY11). Overall, the healthy coronary artery cells were much more homogeneous than atherosclerotic plaque and wall cells. Wall cells (8,224) in atherosclerotic arteries expressed ~no (0.1%) endothelial, many smooth muscle, and fibroblast genes. Plaque cells (8,049) expressed ~no (0.1%) endothelial genes, robust smooth muscle marker genes, and a high abundance of early marker genes for chondrocytes, osteoblasts, monocytes, neutrophils, and macrophages.ConclusionProfound gene expression patterns were shown with scRNAseq in a clinically relevant model of coronary atherosclerosis. The striking heterogeneity of gene expression and marker genes for chondrocytes and osteoblasts in plaque cells indicate significant transdifferentiation and suggest further progression to severe coronary artery calcification.
Impaired Oxygenation of the Right Ventricle during Development of Pulmonary Hypertension in Swine is not due to Loss of Nitric Oxide
Authors: Merkus Daphne , Steenhorst Jarno J. , van Duin Richard W. , Cai Zongye , Duncker Dirk J. 
Published: April 2020

BackgroundPulmonary hypertension (PH) increases workload of the right ventricle (RV), resulting in RV hypertrophy. Eventually the RV is unable to cope with the high afterload, resulting in RV failure and death. The transition of RV hypertrophy to failure is incompletely understood, but we have recently shown that myocardial oxygen supply did not increase commensurate with the increase in myocardial oxygen demand. This mismatch between oxygen supply and demand is exacerbated during physical activity when workload and hence oxygen demand increases. Here, we aim to investigate whether the contribution of the vasodilator nitric oxide (NO) and/or the vasoconstrictor endothelin (ET) to regulation of right ventricular coronary blood flow (RV‐CBF) is impaired in swine with PH.MethodsPH was induced by banding of the confluent of the inferior pulmonary vein in juvenile swine (N=5), while 5 healthy swine served as controls. Swine were chronically instrumented to measure pressure in the RV, pulmonary artery, left atrium and aorta, and right coronary blood flow (RV‐CBF) rest and during graded treadmill exercise. Exercise was performed in the presence and absence of eNOS‐inhibition or ET‐receptor blockade 12 weeks after pulmonary vein banding (PVB).ResultsPVB resulted in PH as evidenced by an increase in pulmonary artery pressure (PVB: 39±3 mmHg, CON: 21±1 mmHg, both P<0.05) that was accompanied by RV hypertrophy (Fulton index PVB: 0.58±0.02, CON: 0.44±0.02, P<0.05). The relation between myocardial oxygen supply (RV‐CBF*arterial oxygen content) and myocardial work (arterial elastance, pulmonary artery pressure/SV) rotated downward in PVB. However, despite increased plasma ET‐levels (PVB:8.8±0.5 pg/ml CON: 6.7±0.5 pg/ml, P<0.05), ET‐receptor blockade did not alter the relation between myocardial oxygen delivery and myocardial work. Conversely, eNOS‐inhibition resulted in a downward rotation of this relation that was more pronounced in PVB as compared to CON.ConclusionPH leads to a mismatch between myocardial oxygen supply and demand. This mismatch is not influenced by ET‐receptor blockade and is aggravated by eNOS‐inhibition, suggesting that NO plays an important role in maintaining RV‐perfusion particularly during exercise.
Autophagy Impairment in Aging Motor Neurons
Authors: Pareja-Cajiao Miguel , Gransee Heather M. , Stowe Jessica M. , Rana Sabhya , Sieck Gary C. , Mantilla Carlos B. 
Published: April 2020

Neuromuscular dysfunction is common in old age. A hallmark of aging is the aggregation of damaged cytoplasmic structures, especially in post‐mitotic cells like motor neurons. Autophagy flux may be impaired as a result of deficits in the formation of double membrane autophagosomes, the fusion of autophagosomes with lysosomes, or lysosome‐based degradation. Our overarching hypothesis is that autophagy contributes to neuromuscular dysfunction in old age, and that this effect occurs in motor neurons. We evaluated changes in expression levels of core autophagy proteins necessary for the initiation (Beclin1) and elongation (LC3‐I, LC3‐II, ATG7 and ATG5‐ATG12 complex) phases of autophagsome formation, as well as lysosome degradation (assessed by p62 clearance), in adult male and female C57BL/6 mice at 6, 18, and 24 months of age (reflecting 100%, 90% and 75% survival, respectively). Protein expression was analyzed by Western blotting in the cervical spinal cord segments C2–C6. Expression of p62 was reduced in the cervical spinal cord of adult mice at 18 months compared to 24 months, suggesting increased clearance and autophagy flux at 18 months of age. There was no evidence of an effect of age on the expression of the other autophagy markers. In separate animals, immunohistochemical (IHC) analyses of the C2‐C6 segments was used to determine the relative expression of LC3 and p62 in gray and white matter regions. Across all age groups, LC3 and p62 immunoreactivity was evident in the gray matter with minimal expression in the white matter. The ratio of both LC3 and p62 in motor neurons vs. gray matter was compared using a mixed linear model with animal as a random effect. No effect of sex was evident. Increased expression of both LC3 and p62 was evident in choline acetyl transferase (ChAT)‐positive motor neurons (~2–3 fold) compared to the remainder of the gray matter. There was evidence of a difference in relative LC3 and p62 expression in motor neurons across age (F349,2 =12.5, p<0.001), with a significant increase by 24 months of age (p<0.005). Taken together, these findings suggest that 1) there is an aging effect on autophagy markers in the cervical spinal cord of mice; 2) an increase in autophagy by 18 months of age reflects an increase in autophagosome clearance (reduced p62 expression) not restricted to motor neurons; and, 3) increased motor neuron expression of both LC3 and p62 by 24 months of age reflect autophagsome accumulation and autophagy impairment in motor neurons. These findings are also is in accordance with previous reports suggesting a period of vulnerability between 18 and 24 months of age in mice, where autophagy may play a role in the development of age‐related neuromuscular dysfunction.Support or Funding InformationSupported by NIH R01 AG057052.
K2P Channel Inhibition Enhances Allergen‐Induced ATP Release by a Mechanism Involving PKC Activation in Human Bronchial Epithelial Cells
Authors: Srisomboon Yotesawee , Maniak Peter J. , Kita Hirohito , O'Grady Scott M. 
Published: April 2020

Allergens derived from the common fungus Alternaria alternata induce oxidative stress in human airway epithelial (HBE) cells that leads to the regulated release of ATP and subsequent activation of P2X7 receptors that facilitate Ca2+ uptake into the cell. Sustained increases in [Ca2+]i are required for nuclear processing and secretion of IL‐33, which initiates allergic inflammation by activating ST2 receptors on immune cells to induce Th2 cytokine secretion. Pretreatment of HBE cells with the K2P channel blockers bupivacaine or quinidine produced a significant increase in ATP release following Alternaria stimulation. This response was blocked by inhibiting ATP loading into membrane vesicles by treatment with 200 nM bafilomycin A1 before allergen exposure. Similarly, pretreatment with the PKC inhibitor Go6976 also blocked approximately 50% of Alternaria‐induced ATP release and completely abolished the increase in ATP release produced by pretreatment with K2P channel blockers. The effects of bupivacaine and quinidine were comparable to the effect of phorbol 12‐myristate 13‐acetate (PMA, 2 mM), a well‐characterized PKC activating compound prior to Alternaria stimulation. These results support the conclusion that K2P channel inhibition is associated with PKC activation, which functions to potentiate the effects of Alternaria allergens on vesicular ATP release. We propose that enhanced PKC activity is dependent on membrane depolarization associated with inhibition of constitutive, K2P channel mediated K+ efflux from these cells.Support or Funding InformationThis study was supported by grants from the NIH (R01‐AI128729), awarded to HK and SMO and from the Minnesota Agriculture Experiment Station (AES‐0016097) awarded to SMO.
Dynamic formation of the malate dehydrogenase/citrate synthase multienzyme complex
Authors: Obata Toshihiro , Krassovskaya Inga , Horová Magdaléna 
Published: April 2020

The enzymes of Krebs tricarboxylic acid (TCA) cycle form multi‐enzyme complexes mediating metabolite (substrate) channeling. As metabolite channeling can enhance the pathway reactions, the multi‐enzyme complexes are postulated to regulate metabolic flux by dynamic association and dissociation. Especially, the interaction between malate dehydrogenase (MDH) and citrate synthase (CS) is expected to have significant impact on the TCA cycle flux since the forward reaction of MDH to produce oxaloacetate from malate is thermodynamically infeasible with physiological metabolite concentrations. However, the dynamics of this multi‐enzyme complex and its relationship with metabolic pathway fluxes have not been demonstrated in vivo. To this end, plate reader‐based split‐luciferase assay was developed to monitor the dynamics of the protein‐protein interaction in living yeast cells and was applied to observe the dynamics of MDH/CS multi‐enzyme complex during the transition of metabolic status. The sequences coding N‐ and C‐ terminus of highly sensitive NanoLUC luciferase were introduced into the yeast genome by homologous recombination to fuse these peptides to the C‐terminus of CS (CIT1) and MDH (MDH1) enzymes, respectively. The resulted yeast strain showed decent fluorescent signals while wild‐type cells showed no background signal. The substrate concentration and cell concentration in the culture were optimized to obtain constant signals for 120 min of measurement. Using this system, the interaction of MDH1/CIT1 complex was monitored following the changes of growth condition which are expected to induce the alteration in the TCA cycle metabolic flux. In the preliminary results, we observed the alteration of luminescence signal in some conditions which are known to modulate the TCA cycle flux. We are currently testing various conditions to analyze the relationship between the dynamics of the multi‐enzyme complex and metabolic fluxes in the TCA cycle and adjacent pathways. We are also trying to identify the factors affecting the interaction of the enzymes.Support or Funding InformationThis study is supported by National Science Foundation, Faculty Early Career Development Program 1845451 to T.O.
Therapeutic anti‐P2X4 receptor scFv antibody for chronic neuropathic pain
Authors: Kunamneni Adinarayana , Montera Marena , Suri Nikita , Westlund Karin N , Alles Sascha R , Pappu Suguna , Durvasula Ravi 
Published: April 2020

The P2X purinoceptor 4 (P2X4) plays a crucial role in signaling leading to chronic inflammatory and neuropathic pains, thus, the P2X4 receptor provides a new potential therapeutic target for their treatment. We have generated a panel of mouse single‐chain Fv‐antibodies (scFvs) recognizing extracellular peptide of P2X4 using cell‐free ribosome display, for use to pain‐signalling neurons. A mouse scFv library was generated, and ribosome display was applied for anti‐P2X4 scFv recombinant antibody selection. After three rounds of bio‐panning, a panel of recombinant antibodies was isolated followed by ELISA, cross reactivity analysis, western blotting and immunofluorescence staining. Anti‐P2X4 scFv clones with high specificity and affinity were successfully selected. Indirect ELISA assay revealed differential P2X4 peptide binding capability and specificity for three scFvs, with no reaction to the negative control anti‐Zika scFv 7‐2. The panning process was efficient in selecting clones of high affinity, as was evident from affinity differences of the scFvs: scFv95 and scFv12 had the highest and second highest affinities, respectively, while scFv103 had lower affinity. The highest affinity P2X4 receptor scFvs (12 and 95) were selected for in vivo behavior testing in the Foramen Rotundum Inflammatory Constriction Trigeminal Infraorbital Nerve injury (FRICT‐ION) Model. Single dose intraperitoneal injection (4 mg/kg, 100 μl) reversed mechanical allodynia and anxiety in mice with long standing neuropathic pain (8–10 weeks). Reversal to naïve baseline within one week was maintained for more than 10 weeks post‐treatment at experiment end. Continued reversal was maintained at baseline for >20 weeks in a second cohort. The Western blot of the medullary brainstem provides evidence of brain penetration of both these scFv antibodies that reduce hypersensitivity and anxiety behaviors. We also performed whole‐cell patch clamp recordings from dissociated trigeminal ganglia neurons of nerve‐injured vs naïve mice and determined the effect of P2X4 receptor scFvs on neuronal excitability. These data provide support for pursuit of P2X4 receptor scFvs as translational therapy for pain relief.Support or Funding Information1. GRANT FUNDING NIH NIDCR R21 DE028096 2. Start‐up Funds to Dr. Durvasula 3. Startup Funds to Dr. Westlund