bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Light‐gated Integrator for Imaging Kinase Activity in Living Cells
Authors: Lin Wei , Phatarphekar Abhishek , Greenwald Eric C. , Zhang Jin 
Published: April 2020

Kinase signaling pathways regulate many cellular processes and are often dysregulated in cancers. The intratumoral heterogeneity of kinase activity in the context of tumor microenvironment presents a challenge for kinase‐targeted cancer therapeutics. Therefore, characterizing kinase activity profiles in complex tumor environment can help address this challenge. Current genetically encoded fluorescent biosensors have been widely used in kinase signaling research. Even with the sensitive and dynamic readout of these biosensors, it is difficult to capture transient changes across a whole tumor section. Herein, we developed a light‐gated
kinase
activity
coupled
transcriptional (KINACT) integrator, which has the capability of “memorizing” differences in kinase activities over a certain period of time. The KINACT integrators have been applied for imaging PKA, PKC and Fyn activities in live cells, as well reporting subcellular changes in PKA activity. Moreover, we applied the PKA integrator to evaluate PKA response to a panel of G‐protein coupled receptor agonists and different kinase inhibitors, suggesting that KINACT integrator is a potential platform for screening various small molecule drugs. The KINACT integrator can also be applied to monitor PKA activity enhancement under pro‐inflammatory conditions.Support or Funding InformationThis work was supported by the National Institutes of Health (R35 CA197622 and R01 DK073368 to J.Z.)
The Mechanism of Actin Remodeling by the BAR Domain of ArfGAP ASAP1
Authors: Gasilina Anjelika , Luo Ruibai , Randazzo Paul 
Published: April 2020

The actin cytoskeleton plays an important role in cellular processes. Normal processes, such as differentiation, use the actin cytoskeleton as a signaling and transport platform, while aberrant processes, such as cancer, repurpose actin for invasion. Actin remodeling involves assembly of globular actin into actin filaments and further assembly of filaments into bundles, which form higher order structures such as stress fibers, lamellipodia and filopodia. A given higher order actin structure relies on organized, timely and regulated mobilization of actin filaments and accessory actin binding and bundling proteins. For one of such structures, the actin stress fibers (SFs), assembly, maturation and maintenance depends on a carefully orchestrated crosstalk between actin filaments, actin binding proteins and the focal adhesion machinery. As a biological phenomenon, SFs provide a mechanotransduction platform, and as a model system, they provide a prototype of the mechanism and the regulation of actin filament assembly. The complexity and diversity of actin structures is, in part, dictated by the actin regulating proteins. Among these are well established actin remodeling families, such as actinin and fascin protein families. More recently a subset of BAR‐domain proteins, widely studied for inducing and sensing the membrane curvature, have been found to control actin filament assembly. However, the mechanism by which BAR domain proteins regulate of the actin cytoskeleton remains unclear. We recently showed that ASAP1, a BAR‐domain containing ArfGAP, remodels actin filaments. Depletion of ASAP1 led to reduction in overall levels of filamentous actin and the number of ECM‐interacting stress fibers. Our structure‐function studies using in vitro actin sedimentation, fluorescence microscopy, as well as cellular assays revealed that the BAR‐PH region of ASAP1 is sufficient and necessary to induce actin reorganization. During the study we observed that another member of the human ArfGAP family that also contains a BAR domain, ACAP1, does not induce the same phenotype as ASAP1. Our current work thus inquires whether structural differences between the BAR domains of ASAP and ACAP explain their disparate effects on actin organization. Molecular modeling‐mediated dissection of the mechanism of actin binding localizes the actin binding site to the 1st and 2nd helix of the BAR domain of ASAP1. Together these data highlight selectivity of the BAR domain proteins in actin remodeling and provide further insights into the different roles human ArfGAPs play in modulating the actin cytoskeleton.Support or Funding InformationThis work was supported by the Intramural Research Program of the National Cancer Institute, Department of Health and Human Services, project number BC007365
ShRNA‐mediated gene silencing of t‐PA prevents BBB disruption and elevation of MMP‐12 after ischemic stroke
Authors: Nalamolu Koteswara Rao , Challa Siva Reddy , Mohandass Adithya , Mussman Justin P. , Ilahi Sadia B. , Bedadala Mayurnath Reddy , Klopfenstein Jeffrey D. , Pinson David M. , Wang David Z. , Kalyanasundaram Ramaswamy , Vemuganti Raghu , Veeravalli Krishna Kumar 
Published: April 2020

Post‐stroke induction of endogenous tissue‐type plasminogen activator (t‐PA) is known to promote BBB disruption after ischemic stroke, which contributes to edema, inflammation, hemorrhagic transformations and brain damage. We presently evaluated if this effect of t‐PA leads to the elevation of MMP‐12 in the ischemic brain. Cohorts of adult, male rats were administered with plasmids that express either a t‐PA shRNA or a scrambled control shRNA (1 mg/Kg; i.v.; n=9–13/group) formulated as nanoparticles at 5 min of reperfusion following transient focal middle cerebral artery occlusion. We also have sham‐operated control and untreated ischemia‐induced cohorts. As compared to control shRNA cohort, t‐PA shRNA treated cohort prevented the degradation of tight junction proteins claudin‐5 and ZO‐1, curtailed BBB disruption (studied by Evans blue extravasation into brain), and showed significantly reduced MMP‐12 expression. These results indicate that post‐stroke induced endogenous t‐PA mediated BBB disruption contributes to elevation of MMP‐12 in the ischemic brain.Support or Funding InformationThis work was supported by the NIH Grant 1R01NS102573‐01A1 to KKV.
Switching hypoxia tolerance strategies in a brainstem network of bullfrogs, Lithobates catesbeianus
Authors: Adams Sasha , Santin Joseph M. 
Published: April 2020

Strategies to survive brain anoxia vary widely among vertebrates. Along a spectrum, some animals arrest neuronal activity to conserve ATP, while others can sustain neuronal functions during energetic stress. The neural circuit that produces breathing in the adult American bullfrog, Lithobates catesbeianus, is one example of a network that arrests activity during anoxia. This response is considered an energy saving arrest because 1) a hypoxia sensor that inhibits the network through noradrenergic signaling appears to contribute, and 2) activity recovers after anoxia. Unexpectedly, we found that this network becomes remarkably functionally tolerant to hypoxia after frogs emerge from 3–5 weeks of cold‐acclimation. Normally, control networks fail to produce rhythmic activity after 10.1±6.4 minutes of hypoxia (0% O2) at 22°C (n=9); however, networks at 22°C from cold‐acclimated frogs continue to produce coordinated respiratory bursts for about 16 times longer (159.1 ±42.2 minutes; n=5; p=0.001; Mann‐Whitney U test), representing a shift from an arrest strategy to functional tolerance. Thus, the transition may reflect a reduction in the hypoxia sensor (noradrenergic signaling) and/or a shift in energy use/metabolism. To begin to address these possibilities, we sought to identify if noradrenergic signaling is the sole cause of the arrest in control networks. To test this, we exposed in vitro brainstem preparations to norepinephrine and then hypoxia. We expected that pretreatment with norepinephrine would occlude the arrest during hypoxia if noradrenergic signaling was part of the oxygen sensor. However, this did not occur; preparations stopped activity over a similar time course during hypoxia when bathed with (n=6) or without (n=9) norepinephrine, implying the arrest does not involve noradrenergic signaling. We then hypothesized that metabolic deficits may contribute. To test this, we applied inhibitors of glycolysis [2‐deoxy‐D‐glucose substituted for D‐glucose (n=5) or 1 mM iodoacetate (n=4)] or mitochondrial respiration [1 mM sodium cyanide (n=5)] in baseline O2 tensions. Each inhibitor recapitulated the actions of hypoxia, demonstrating that deficits in ATP synthesis, regardless of oxygen tensions, produce an arrest. Cellular energy sensors, such as KATP channels and AMP‐dependent protein kinase, are known to reduce neuronal activity when ATP falls; however, activation of these mechanisms pharmacologically failed to arrest activity [KATP activator; 600 μM Diazoxide (n=5], AMPK activator; 1 mM AICAR (n=4)]. In sum, we hypothesize that deficits in ATP synthesis stop network output during hypoxia and that improvements in metabolic function or energy use contribute to the striking shift to functional hypoxia tolerance after cold‐acclimation.Support or Funding InformationThis work was supported by start‐up funds to JS provided by UNC‐Greensboro.
The ERG1 Potassium Channel is Abundant in Cachectic Human Skeletal Muscle
Authors: Pond Amber Lynn , Zampieri Sandra , Sandri Marco , Cheatwood Joseph , Kohli Punit , Balaraman Rajesh , Anderson Luke Brian , Latour Chase Doyne , Hockerman Gregory H. , Kern Helmut , Sartori Roberta , Merigiano Stefano , Da Dalt G , Davie Judy K. , Carraro Ugo 
Published: April 2020

IntroductionA heteromultimer of the ERG1a/1b potassium channel is known to contribute to repolarization of the cardiac action potential. A homomultimer of the ERG1a subunit has been detected in the atrophying skeletal muscle of mice experiencing muscle disuse and cancer cachexia and has been shown to contribute to muscle atrophy by enhancing ubiquitin proteolysis; however, to our knowledge, ERG1 has not been reported in human skeletal muscle.Methods and ResultsHere, using immunohistochemistry we detect ERG1 immunofluorescence at low levels in Rectus abdominis muscle of young adult humans and show that it trends toward greater levels (10.6%) in the same muscle of healthy aged adults. Further, we detect ERG1 immunofluorescence at a statistically greater level (53.6%; p<0.05) in the Rectus abdominis muscle of older people having cancer cachexia than in age‐matched adults. Additionally, we observe ERG1 immunofluorescence in skeletal muscle sarcolemma and detect that its fluorescent pattern is consistent with I‐band localization.DiscussionThe data suggest that ERG1 may be related to muscle loss in humans and may be located in t‐tubules where it could influence calcium handling.Support or Funding InformationThis work was supported in part by Southern Illinois University School of Medicine (Research Seed Grant to ALP). The work was also supported in part by the Italian Society for Cancer Research (AIRC grant number 17388 to M Sandri) and by Austrian national co‐financing of the Austrian Federal Ministry of Science and Research; Ludwig Boltzmann Society (Vienna, Austria) to H Kern. PK would like to acknowledge National Science Foundation (CHE 0748676), NIH (GM 106364) for partial financial support of this research. The authors wish to thank Dr. Don Caspary who graciously provided FBN rat muscles. Human Pectoralis minor muscle samples were provided by the SIU School of Medicine Tissue Bank, a Simmons Cancer Institute funded program. Other investigators may have received samples from these same tissue specimens. UC thanks the IRCCS Fondazione Ospedale San Camillo, Venezia, Italy and the A&C M‐C Foundation for Translational Research for scientific support.
Microglia regulate neuronal network formation and synaptogenesis
Authors: Ruß Tamara Heike , Spittau Björn , Piecha Robin 
Published: April 2020

Microglia are the resident immune cells of the central nervous system (CNS) and play an essential role under physiologic and pathologic conditions. Next to their well‐established role in the progression in neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD), recent reports underline the involvement of microglia during postnatal CNS maturation. The main objective of the present study was to examine the influence of microglia on neuronal network formation and synaptogenesis in cortical/midbrain neuron cultures.Primary mouse E14 cortical/midbrain neuron cultures were used to determine the development of functional synapses in the presence and absence of microglia. Microglia depletion was performed using the Csf1r inhibitor BLZ945. Neuronal network formation and maturation of synapses were analyzed using the Microelectrode Array Technology (MEA) and immunocytochemistry labeling of synaptic markers such as Synapsin and PSD95.Electrophysiology using MEAs indicated that microglia regulate the abundance of functional synapses during a time of 28 days in vivo (DIV). Using a Csf1r inhibitor, we were able to show that the efficient depletion of microglia abrogated the inhibitory effects on synaptogenesis.Taken together, our data demonstrate that microglia are effective regulators of neuronal network formation and synaptic connectivity further broadening the portfolio of versatile functions of these resident immune cells of the central nervous system.
Identification of Residues Controlling dUTPase Enzyme Activity from Epstein‐Barr Virus
Authors: Robbins Alex M. , Gorres Kelly 
Published: April 2020

Epstein Barr‐Virus (EBV) is primarily known for causing infectious mononucleosis, but also increases the incidence of lymphomas and autoimmune disorders. There is currently no vaccine or treatment once infected by EBV. The EBV genome encodes a dUTPase enzyme. This enzyme catalyzes the reaction of dUTP to dUMP which is a precursor of the thymidine nucleotide. The enzyme activity regulates the dUTP/dTTP ratio and decreases misincorporation of uracil into newly synthesized DNA. A dUTPase knockout is lethal in mice, E. coli., and S. cerevisiae. The Murine gammaherpesvirus lacking its viral dUTPase has reduced viral replication. It is hypothesized that inhibition of EBV dUTPase would result in uracil misincorporation, double stranded DNA breaks, and a reduction in replication of the viral genome. Characterization of residues required for enzyme activity in EBV dUTPase was performed using sequence alignments and structural data. Mutagenesis and comparative enzyme kinetics on these residues identified aspartate 76 as being critical for enzyme activity. Analysis of structural data identified a histidine residue hypothesized to regulate substrate specificity. A small molecule binding site was identified using computational mapping of two EBV dUTPase 3D structures. We hypothesis that this allosteric binding site contains a serine that post transcriptionally regulates dUTPase activity. This research will guide the development of inhibitors that bind to critical dUTPase residues offering a potential treatment for EBV.Support or Funding InformationThis work was supported by, UWL College of Science and Health, UWL Research and Creativity, and Toce Deans Distinguished Fellowship
Investigating Plasma Membrane Monoamine Transporter (PMAT) as a Target for Development of Novel Antidepressant Treatments for Juveniles
Authors: Bowman Melodi Ann , Vitela Melissa , Gilman T. Lee , Daws Lynette C. 
Published: April 2020

Depression is a psychiatric illness that affects individuals of all ages, yet only two antidepressants are approved to treat depression in children – both belong to the selective serotonin reuptake inhibitor (SSRI) class. Moreover, children are less effectively treated by SSRIs than adults. SSRIs block reuptake of serotonin via the high‐affinity, low‐capacity serotonin transporter (SERT). The resulting increase in extracellular serotonin is thought to initiate a cascade of downstream effects, which underlie the therapeutic utility of SSRIs. However, other transporters also clear serotonin from extracellular fluid, including the low‐affinity, high‐capacity organic cation transporters (OCTs) and plasma membrane monoamine transporter (PMAT). Our lab has shown that, in adults, decynium‐22 (D22), an inhibitor of OCTs and PMAT, produces antidepressant‐like effects when SERT function is either genetically or pharmacologically impaired. However, it is unknown whether OCTs and/or PMAT may be useful targets for therapeutic interventions in juveniles. In contrast to adults, our preliminary studies show that D22 has antidepressant‐like effects in juvenile (postnatal day 21 (P21)) wild‐type mice, suggesting that OCTs and/or PMAT may be functionally upregulated in juvenile mice. Since PMAT has a higher affinity for serotonin compared to OCTs, we investigated the antidepressant‐like effects of D22 in male and female P21 PMAT wild‐type (+/+) and constitutive PMAT knockout (−/−) mice and found the antidepressant‐like effects of D22 were lost in P21 PMAT−/− mice. Using in vivo chronoamperometry, we examined serotonin clearance kinetics in CA3 region of hippocampus in P21 PMAT+/+ and PMAT−/− mice. Across serotonin concentrations ranging from 0.25 to 1.5 micromolar, we found no difference in serotonin clearance rate between genotypes. These data suggest that either a) these concentrations of serotonin are insufficient to engage PMAT, and/or b) SERT, or other OCT isoform, is upregulated in constitutive PMAT−/− mice. In either case, detection of PMAT‐dependent serotonin clearance would be obscured. Ongoing studies are investigating the effect of D22 on serotonin clearance in juveniles and adults over a range of serotonin concentrations, as well as evaluating PMAT expression in brain as a function of age and PMAT genotype. Together, these studies will expand our understanding of differences in serotonin transport mechanisms between juvenile and adult brain, and will aid in discovery of novel targets for the development of antidepressants with improved therapeutic efficacy for children suffering from depression.Support or Funding InformationThis work was supported by the National Institutes of Health R01 MH106978 to LCD
Bioelectrical Impedance Analysis in Breastfeeding Women After an Individualized Dietary Intervention
Authors: Trecierras Larisa , Limon-Miro Ana T. , Valencia Mauro E. , Plata Trinidad Quizan , Slater Christine , Guzman-Leon Alan E. , Padilla Andrea Garcia , Valenzuela Jose C. , Lopez-Teros Veronica 
Published: April 2020

Body composition assessment by bioelectrical impedance analysis (BIA) in breastfeeding women can provide an insight on the changes in body tissues and the state of hydration. Our general aim was to assess the changes of BIA parameters in overweight or obese breastfeeding women after an individualized nutritional intervention. After an informed consent was signed, breastfeeding women (2–8 weeks postpartum) were randomly assigned by simple allocation in blocks (1:1) to either the intervention (In) or control (Cn) group. These two groups received general recommendations on healthy eating during the lactation period; additionally, the In group received a dynamic menu with meals based on equivalent interchangeable foods every 2 weeks for 3 months. Body composition was evaluated using anthropometry and multi‐frequency bioelectrical impedance (Impedimed™) at baseline and 3 months after the intervention. Out of 345 women invited, 37 were eligible but only twenty (n = 10/group) completed the intervention program. At baseline, there were no differences in age and body mass index (BMI) and 60% of both groups of women declared exclusive breastfeeding. At the end of the program, the In group showed differences of −2.9 kg of fat mass [IQR −7.1– −0.9 (p<0.05)], −2.5 cm in waist [(IQR −5.0 – 0.2 (p=0.06)] and −3.2 cm in hip circumferences [IQR −4.7– −1.7 (p<0.05)]. Change in resistance standardized by height was ‐ 27 ohms [IQR −63–0.7 (p<0.05)]. Fat free mass and total body water remained unchanged with medians of +1.4 kg [IQR −0.15–2.6 (p=0.4)] and +0.95 kg [IQR −1.5– 3.2 (p=0.1)], respectively. This individualized nutrition intervention program designed for breastfeeding women was effective in improving the mother’s body composition. Multi‐frequency BIA can be a useful tool to monitor these changes during postpartum subjects in clinical practice. This trial was registered at clinicaltrials.gov NCT03640104.Support or Funding InformationLT, AGP, JCV recieved a fellowship from The National Research Council (CONACYT)
Protein Arginine Methyltransferase 7 (PRMT7): A Human Enzyme Often Overexpressed in Cancer is Most Active Under Non‐physiological Conditions
Authors: Lowe Troy L. , Vernon Annabel , Clarke Steven G. 
Published: April 2020

Arginine methylation is a ubiquitous eukaryotic posttranslational modification that can modulate transcriptional activation and suppression. Protein arginine methyltransferase 7 (PRMT7), one of the nine mammalian PRMT family members, catalyzes only monomethylation of a terminal guanidino nitrogen atom on its substrates. In an effort to understand the role of PRMT7 in the cell, we characterized a recombinant human PRMT7 expressed as a GST‐fusion protein in bacterial cells. Using S‐adenosyl‐L‐[methyl‐3H]methionine (3H‐AdoMet) as a substrate, we measured the rate of methylation of a synthetic peptide containing residues 23–37 of human histone H2B. Although the physiological methyl‐accepting substrates of PRMT7 are not known, this peptide contains major in vitro monomethylation sites at arginine residues 29, 31, and 33. A P81 phosphocellulose binding assay was performed that separates the 3H‐methylated product from unused 3H‐AdoMet. We confirmed the unusual temperature dependence of PRMT7, where optimal activity was found at about 20 °C with only about 30% of this activity at 37 °C. We found that the Km of PRMT7 for the H2B‐derived peptide was about 10 μM at 20 °C and about 20 μM at 37 °C. The Km for AdoMet was about 0.5 μM at both temperatures. Additionally we showed that physiological salt conditions can significantly decrease the activity. At 150 mM concentrations, we found only about 10% activity with NaCl or KCl, but about 80–90% activity with potassium acetate or ammonium acetate. We also detected a loss of activity with high ionic strength. These results suggest that there may be a specific inhibition by chloride ion as well as by ionic strength. Finally we show that PRMT7 activity is optimal in alkaline environments. The activity of PRMT7 at the near‐physiological pH of 7.5 is only about 20% that at pH 8.5. Taken together, these results open the possibility that PRMT7 can respond to cells undergoing temperature, salt, or pH stress. These findings demonstrate a potential mechanism for the in vivo regulation of arginine methylation by PRMT7 and provide clues to why PRMT7 has pleiotropic effects involved with transcriptional regulation and translation in the cell.Support or Funding InformationThis work was supported by National Science Foundation Grant MCB‐1714569 (to S.G.C), by a predoctoral Ruth L. Kirschstein National Service Award GM007185 (to T.L.L), and by an Amgen Scholars Award (to A.V.).