bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of PAR‐1 Receptor Inhibition in an In Vitro 6‐hydroxydopamine Model of Parkinson’s Disease Using Differentiated PC12 Cell Line and Undifferentiated SH‐SY5Y Cell Line.
Authors: Natu Kalyani Narendra , Migliore Mattia M. 
Published: April 2020

Parkinson’s disease (PD) is a progressive neurodegenerative condition affecting dopaminergic neurons of the A9 nigrostriatal pathway. Besides the hallmark features of bradykinesia, cogwheel rigidity, and resting tremor, PD patients are at an increased risk of strokes. Protease‐activated receptors (PARs) and thrombin have been implicated in the neuronal damage following both ischemic and hemorrhagic strokes, and may play a role in the neurodegeneration observed in PD. Furthermore, brain PAR receptor expression appears to be upregulated in PD patients. The purpose of this study is to determine if PAR‐1 receptor inhibition can provide neuroprotection in two commonly used in vitro 6‐hydroxydopamine (6‐OHDA) models of PD: differentiated PC12 cells vs. undifferentiated SH‐SY5Y cells. In a previous study, we demonstrated an antiproliferative effect on the A549 cancer cell line via inhibition of PAR‐1 receptors with enoxaparin. We hypothesize that PAR‐1 receptor inhibition will result in neuroprotection from 6‐OHDA toxicity in differentiated amitotic PC12 cells and will promote neurotoxicity in undifferentiated SH‐SY5Y cells. This study hopes to elucidate the mechanisms behind this differential effect of PAR‐1 receptor inhibition in the two cell lines. To this end, Western Blot analysis was conducted to confirm that PAR‐1 receptors were expressed in differentiated PC12 cells and undifferentiated SH‐SY5Y cells. Then, both cell lines were treated with a potent PAR‐1 antagonist, SCH79797 dihydrochloride (SCH), and cell viability was assessed by the MTS Assay. PAR‐1 blockade did in fact have a differential effect in the two cell lines by reducing the cell viability of undifferentiated SH‐SY5Y cells, while having no effect on the cell viability of differentiated PC12 cells. Consequently, both the cell lines were exposed to 6‐OHDA and differentiated PC12 cells were treated with SCH after inducing toxicity with 6‐OHDA. Preliminary results suggest that the selective PAR‐1 antagonist, SCH79797 dihydrochloride, may provide a protective effect against 6‐OHDA toxicity in differentiated PC12 cells. Additional studies will be conducted to determine the effect of SCH on 6‐OHDA treated undifferentiated SH‐SY5Y cells, and the mechanisms involved in this differential effect of PAR‐1 receptor inhibition.Support or Funding InformationDepartment of Pharmaceutical Sciences, MCPHS University, Boston, MA.PAR‐1 Receptor Inhibition Increases Cell Viability in a PC12 model of Parkinson’s Disease:Effect of treatment with SCH79797 dihydrochloride (SCH; 1 μM) on differentiated PC12 cells exposed to 6‐hydroxydopamine (6‐OHDA; 250 μM) toxicity as assessed by the MTS Assay. Data is represented as a mean ± S.D. of three independent experiments (n=3). * p<0.0001 as compared to the untreated control, # p<0.005 as compared to the model group.Figure 1
Greater Angiotensin II Metabolism in Rodents Compared to Humans: Role of Aspartyl and Dipeptidyl Aminopeptidases
Authors: Chappell Mark C. , Pirro Nancy T. 
Published: April 2020

IntroductionFunctional tone of the renin‐angiotensin system (RAS) is influenced by the balance of Angiotensin II (Ang II) generation and metabolism. The major if not sole Ang II‐forming enzyme is ACE while amino (AP)‐, endo (EP)‐ and carboxy (CP)‐peptidases degrade Ang II. Rodents are the primary models for Ang II‐evoked hypertension, inflammation and fibrosis, and genetic hypertensive models typically overexpress various RAS components that stimulate the ACE‐Ang II‐AT1 receptor axis. The present study compared Ang II degradation and generation pathways in humans and rodents. We hypothesize that significant species differences regarding Ang II metabolism may exist among humans and rodents.ResultsRate of Ang II metabolism in serum (5.0 μl at 37°C for 4 hours) was 9‐fold higher in Sprague‐Dawley rats and 2‐fold higher in C5Bl/6 mice than humans (Fig.
1A). In rats, Ang II metabolism was AP‐dependent forming Ang‐(2‐8), Ang‐(3‐8) and Ang‐(4‐8). AP inhibitors bestatin and amastatin (10 μM each) abolished Ang‐(3‐8) and Ang‐(4‐8), but increased Ang‐(2‐8). Cystiene peptidase inhibition (PCMB) blocked Ang‐(2‐8) generation that distinguished Aspartyl‐AP (Asp‐AP) from Glutamyl‐AP (Glu‐AP) in serum. Complete AP blockade abolished Ang II metabolism in rats. In mice, Ang II metabolism was primarily mediated by a dipeptidyl‐AP(DAP) and to a lesser extent by Asp‐AP and Glu‐AP following DAP blockade. Ang II generation was 2–3‐fold higher in mice than rats and humans (Fig.
1B). Finally, there was no evidence of Ang‐(1‐7) formation from Ang I or Ang II in the serum of all three species.ConclusionTo our knowledge, this is the first study that established marked differences in Ang II metabolism between humans and rodents that reflects substantially higher Asp‐AP in rats and greater DAP‐3 in mice. The greater AP and DAP activities may constitute a cardioprotective mechanism particularly in the rat, although additional studies are required to determine whether other tissue compartments express such dramatic differences in AP activity and Ang II metabolism.Support or Funding InformationAHA Transformative Grant 18TPA34170522, AHA 14GRNT20480131; NIH 1R01HL146818, NIH HD‐084227;Ang II metabolism (A) and Ang II formation (B) in rat, mouse and human serum. *P<0.05; n=3 determinations from a pool of 10 each.Figure 1
Integrated 3D Tomography and Computational Modeling to Study Forces in Metaphase Spindles
Authors: Redemann Stefanie , Nazockdast Ehssan 
Published: April 2020

Chromosome segregation during mitosis and meiosis are fundamental processes of life. During its course a bipolar spindle assembles and initially aligns the chromosomes on a metaphase plate before they are segregated to the two arising daughter cells during anaphase. The different cellular components of mitotic and meiotic spindles: microtubules, chromosomes, motor proteins and also membranes, such as the cell cortex or nuclear envelope, must interact with each other during spindle assembly, chromosome alignment and segregation.While we have achieved a very good understanding of signaling cascades and checkpoints that monitor and control mitosis, we are lacking an understanding on how the nano‐scale components of a spindle physically interact and generate the forces needed to faithfully align and segregate
chromosomes. We have very little knowledge about the mechanics of spindles. In order to overcome the current limitations, we are generating a holistic description of mitotic and meiotic
spindles in C. elegans embryos and mouse oocytes. This ambitious task will be achieved by a combination of 3D electron tomography, light microscopy and computational simulations. 3D tomography provides unprecedented detailed information about every individual microtubule within the spindle. We can measure the length, position and interactions of single microtubules with subcellular targets. In addition, we have developed tools to analyze the curvature of microtubules. Curvature originates from forces experienced by the microtubule and provides a read out of spindle intrinsic forces. The source of those forces can be determined by a combined approach of mutant analysis and computational simulations. To complement the static tomography data, we perform state‐of‐the‐art light microscopy. In conjunction with the light and electron microscopy we develop hypothesis and mechanical models of chromosome congression, alignment and segregation and test those in computational
simulations and by selective depletion of proteins. We are using this approach to develop a (near) true‐to‐life model of microtubule, chromosome and membrane interactions and forces during mitosis.3D reconstruction of a meiotic metaphase spindle in the C. elegans embryoThis reconstruction is generated using 3D electron tomography of high‐pressure frozen samples and subsequent image processing and segmentation. The resolution of 2.3nm provides single microtubule resolution.Figure 1
Lessons from the Lamprey on Spinal Cord Regeneration
Authors: Morgan Jennifer 
Published: April 2020

Unlike mammals, non‐mammalian vertebrates undergo robust, spontaneous regeneration of central nervous system (CNS) structures after injury. Lampreys, which are amongst the most basal vertebrates, provide a unique opportunity to study CNS regeneration at levels ranging from molecules to behavior. After a complete spinal cord transection, lampreys recover nearly normal locomotor behaviors (e.g. swimming, burrowing) over the next 10–12 weeks. This functional recovery is accomplished by robust tissue repair, axon/synapse regeneration, and transcriptional changes in the spinal cord and brain that support CNS repair and growth. Such growth programs are highly conserved across vertebrates, including in mammals. Additionally, lampreys possess a class of large, identified reticulospinal neurons with known regenerative capacities: some are “good regenerators,” while others are “poor regenerators,” allowing us to examine intrinsic factors that govern CNS regeneration on neuron‐by‐neuron basis. This presentation will discuss some of the latest findings in lamprey CNS regeneration, including new molecular strategies with potential for improving CNS regeneration outcomes in other preclinical animal models.Support or Funding InformationNY Spinal Cord Injury Research BoardMarine Biological Laboratory, Woods Hole, MALamprey spinal cord at 11 weeks post‐injury. Many axons (green: neurofilament) are regenerating across the lesion, and the central canal that runs longitudinally down the center of the spinal cord is repaired (blue: DAPI). The original lesion site is in the middle of the image.Figure 1
Novel 2‐Oxoglutarate Analogues Modulate the Epigenetic Activity of the Cancer‐related Human Enzyme Aspartate/Asparagine‐β‐Hydroxylase
Authors: Brewitz Lennart , Tumber Anthony , Nakashima Yu , Schofield Christopher J. 
Published: April 2020

The human oxygenase aspartate/asparagine‐β‐hydroxylase (AspH) catalyzes posttranslational β‐hydroxylations of specific Asp/Asn‐residues in epidermal growth factor‐like domains (EGFDs) by using 2‐oxoglutarate (2OG) as a cofactor.
[1] AspH is overexpressed in certain cancer cells and translocates from the endoplasmic reticulum to the cell surface membrane, a process which correlates to enhanced tumor invasiveness and poor clinical prognosis.
[2] We recently reported the production of a truncated human AspH‐construct in E. coli that was catalytically active
[3] and used for the development of a high‐throughput mass spectrometry‐based assay to monitor AspH‐activity in vitro. Herein, we report a novel synthesis of non‐natural 2OG analogues which were either alternative AspH‐cofactors or small‐molecule AspH‐inhibitors.Commercial starting materials were converted in three synthetic steps to previously inaccessible 2OG analogues bearing alkyl‐, aryl‐, or heteroaryl‐substituents. This novel synthesis compares favorably to literature‐reported syntheses as it features a broad substrate scope, high reaction yields, and simple reaction protocols. A diverse library of 2OG analogues (>35) was obtained and evaluated in the AspH‐assay. Potent AspH‐inhibitors were identified and ranked according to their (IC50)‐values, constituting the first detailed structure activity relationship study on AspH using small‐molecules with a known mode of inhibition (replacing 2OG in the active site). Several 2OG analogues could substitute 2OG as cofactor and thus promote enzymatic turnover. Michaelis constants (KM) were determined and used to compare the efficiency of the 2OG analogues with 2OG. The selectivity of the novel AspH‐inhibitors and ‐cofactors was evaluated using structurally and functionally related human 2OG‐dependent oxygenases (i.e. PHD2, FIH, JMJD5). Finally, crystallographic experiments were performed to obtain further structural information on the substrate requirements of the AspH active site; thus enabling future studies to design selective AspH‐inhibitors as potential anticancer therapeutics.In conclusion, a novel efficient synthesis of 2OG analogues was developed providing access to important chemical tools for mechanistic and inhibition studies of 2OG‐dependent oxygenases. The relevance of this compound class was highlighted by the identification of novel potent and selective small‐molecule inhibitors of the cancer‐related human enzyme AspH.Support or Funding InformationWe thank the Wellcome Trust and Cancer Research UK for funding. L.B. thanks the Deutsche Forschungsgemeinschaft for a fellowship (BR 5486/2‐1).
Dynamic Collapse Mechanism of Alveolar Type 1 Caveolae Buds Modeled as Cubic Mesophase Lyotropic Liquid Crystal Formation and Growth
Authors: Newson Jordan 
Published: April 2020

The alveolar type 1 (AT1) cells cover 95% of the cell surface area while the alveolar type 2 cells cover the remaining 5% of 100–150 m2 surface area of the lung. AT1 maintain membrane integrity by surface tension via caveolin proteins that stretch and collapse the membrane bilayer in physiological pressure cycling, however, collapse mechanics of the AT1 membrane have not been elucidated. Golani et al. modeled globular caveolin collapse structure base on mean curvature and lateral tension, and other sources have constructed similar models. The parameters are transformations of the critical packing parameter CPP equals monomer dimensions predicts that the monolayer shape of surfactants, described as v/al. Deng et al. model for gemini surfactants at the air‐water interface and Turner et al. model for the formation of caveolae bulbs via energy displacement of a membrane bound proteins have proposed similar geometric theories that show resemblance to lyotropic liquid crystal swell equation. The models feature common mathematical properties which are 1) spherical geometry, 2) lattice parameter‐based expansion, and 3) monomer population as mechanical descriptors of bilayer transformation. Each model correlated positively with cubic formation and decrease in surface tension. Krianeva et al. showed that monolayer of monoolein formed a new phase of bicontinuous phase, P4332, as the concentration of protein Horse cytochrome c increased. The spherical formation models predicted mesophases are cubic meaning that they are CPP >> 1, micellar cubic shape. The overall result, the caveolin bud shape is cubic bicontinuous mesophase, which is stabilized under physiological pressure cycling in the AT1 cell. We propose, the caveolin bud shape is a cubic bicontinuous mesophase stabilized under physiological pressure cycling in AT1 cell membrane and mechanics can be modeled by using the lamellar to cubic transition or the swelling law using change in surface tension vs lattice parameter as the formation rate. Furthermore, caveolin and cavin proteins present during the time of formation induce invagination, and, second, stabilization and fragility. Agarwal et al. show the transition phase between isotropic and cubic mesophase is a volume fraction difference of 0.05, a metastable polyhedral lattice produced by caveolin proteins, characterizing bud formation as plastic liquid crystal phase transition. This model could explain mechanoprotection by caveolae bud, and gives us a platform to think about self‐assembling monomer‐based medicines when approaching physical stretch dysfunction repair in the lung.Equations are models that describe cubic expansion from the lamellar setting across several membrane simulations. Equation 1 (left) describes the radius, r1, of Gemini surfactant. Equation 2 (middle) describes the formation of caveolae bulbs via the energy displacement of membrane‐bound proteins. Equation 3 (left) describes the swelling law of lyotropic mesophases.Figure 1
Bladder Enlargement Correlates with Plasma Insulin, Not Glucose Levels in Fructose‐Fed Rats
Authors: Michel Martin C. , Beltran-Ornelas Jesus H. , Silva-Velasco Diana L. , Yesilyurt Zeynep E. , Arioglu-Inan Ebru , Centurion David 
Published: April 2020

IntroductionMajor bladder enlargement is observed in many, but not all rodent models of diabetes (Neurourol Urodyn 37: 2346–2360, 2018). According to one theory, bladder enlargement occurs secondary to increased diuresis, which in turn occurs secondary to high plasma glucose levels. However, comparisons across models show only moderate correlations between bladder enlargement and glucose levels. In a recent analysis, we found that correlations within models are tight in some (e.g. rat insulin promotor lymphocytic choriomeningitis virus mice), but weak to non‐existent in other models (e.g. insulin receptor substrate 2 knock‐out or ob/ob mice). Therefore, we have expanded our investigation to another model, fructose‐fed rats.Methods7‐week old male Wistar rats received tap water or water containing 15% fructose for 20 weeks (n = 6 per group). At the end of treatment, an oral glucose tolerance test with quantification of plasma glucose and insulin levels at various time points was performed; bladder and body weight were determined.ResultsWhile basal glucose and glucose area under the curve (AUC) during the glucose tolerance test were similar in both groups, basal insulin and AUC were increased in fructose fed rats (basal mean difference 0.46 mIU/ml*120 min (95% CI 0.17; 0.75)), indicating insulin resistance. Bladder weight was greater in fructose‐fed rats (mean difference 79 mg (95% CI 56; 102)). Bladder weight did not correlate with basal glucose or AUC, but with basal insulin and, somewhat weaker, AUC (r2 0.5127 and 0.3321).ConclusionsWe propose that insulin rather than glucose levels may be the driver of bladder enlargements in some models of diabetes and hyperinsulinemia.Support or Funding InformationDeutsche Forschungsgemeinschaft Mi 294/8‐1; Conacyt Mexico 252702; Ankara University BAP‐16L0237006; Scientific and Technological Research Council of Turkey TUBITAK SBAG‐115S564 and TUBİTAK‐2211/ACorrelation of bladder weight with glucose and insulin levelsFigure 1
OPIOID‐RESISTANT RESPIRATORY RHYTHM SPONTANEOUSLY RESCUING UN‐ANESTHETIZED RATS FOLLOWING FENTANYL OVERDOSE INDUCED APNEA
Authors: Haouzi Philippe , McCann Marissa , Tubbs Nicole 
Published: April 2020

In un‐anesthetized rats, a slow rhythmic respiratory activity spontaneously resumes within tens of seconds following fentanyl overdose induced apnea, rescuing the animals. As this rhythm is produced at a time wherein fentanyl concentrations and receptors occupancy are likely to be extremely high, the mechanisms initiating and sustaining such a respiratory activity remains very unclear.In an attempt to describe the opioid sensitivity and the chemo‐sensitivity of this “rescuing” neo‐rhythm, twenty‐six un‐anesthetized adult rats were studied in an open‐flow plethysmograph. The animals were acutely intoxicated with a bolus intravenous injection of a very high dose fentanyl (300 μg.kg−1), while being exposed to different gas composition (air, 100% O2, 8% CO2/15% O2). All air breathing rats presented an apnea 16 ± 5 seconds into the onset of fentanyl administration and started to generate rhythmic breaths 93 ± 22 seconds into apnea. Ventilation was significantly depressed during this recovery phase along with V̇E/V̇CO2 ratio. Neither hyperoxia nor hypercapnia affected the time at which a spontaneous respiratory rhythm was produced or the level of ventilation that ensued. At 10 minutes, the animals were randomly divided into 2 groups: one group immediately received a second injection of fentanyl, while the other group received a second dose at 30 minutes. No apneas were produced after a second injection of fentanyl in 100% and 70% of the rats at 10 and 30 minutes respectively.The rhythmic breathing activity that rescues fentanyl overdose‐induced apnea is chemo‐insensitive and opioid resistant for at least 30 minutes. Investigating the mechanisms of desensitization involved in this survival mechanism, which develops within tens of seconds – and whether or not such a mechanism relies on the opioid phosphorylation of G coupled receptors/beta arrestin 2 interactions – could lead to a better understanding of the mechanisms leading to a fatal outcome of fentanyl induced apnea and possibly to the development of new countermeasures against opioid overdose.Support or Funding InformationNONEExample of the effects of injections of fentanyl (300 microg.kg−1.min−1 over one minute) on instantaneous respiratory flow and minute ventilation 10 minutes apart in an unseated rat (open‐flow body plethysmography). A long central apnea occurred almost immediately after the first injection (first bout) followed by the emergence of a spontaneous rhythmic breathing with slow frequency and low tidal volume. A subsequent injection, 10 minutes later, produced almost no change in breathing.Figure 1
The Prolyl Isomerase PIN1 Impairs Methionine Adenosyltransferase α1 Mitochondrial Targeting in Alcoholic Liver Disease
Authors: Torres Lucia Barbier , Murray Ben , Yang Jin Won , Robinson Aaron , Wang Jiaohong , Eyk Jenny Van , Sun Zhaoli , Liangpunsakul Suthat , Mato Jose M. , Lu Shelly C. 
Published: April 2020

BackgroundMethionine adenosyltransferase alpha1 (MATα1) is responsible for hepatic biosynthesis of S‐adenosylmethionine (SAMe). Lower MATα1 activity and SAMe levels occur in alcoholic liver disease (ALD), a condition characterized by mitochondrial dysfunction. We recently showed MATα1 is in the mitochondrial matrix of hepatocytes and is important for mitochondrial function. PIN1 is a peptidyl‐prolyl cis‐trans isomerase that can alter the activity, stability, interactions and subcellular localization of its targets by binding to a specific phosphorylated motif pSer/pThr‐Pro. PIN1’s role in ALD is unknown and MATα1 has a PIN1 binding motif (Ser114‐Pro115). The aim of this study was to examine whether PIN1 can regulate MATα1 function in ALD.MethodsMATα1 phosphorylation was examined using immunoprecipitation (IP) with anti‐phospho‐Ser antibody followed by immunoblotting, phosphoproteomics and mass spectrometry (MS). IP using recombinant proteins, liver and cell lysates studied MATα1‐PIN1 interaction. Mitochondria were isolated from AML12 cells treated with 100mM ethanol for 48 hours (in vitro model), NIAAA livers (in vivo model), liver samples from patients with alcoholic hepatitis, and HepG2 cells where PIN1 expression was varied to evaluate MATα1 mitochondrial content. MATα1 S114A mutation investigated its impact on PIN1 interaction.ResultsPIN1 expression was unchanged in ALD models and in the livers of ALD patients. We found that MATα1 and PIN1 interact directly, in liver and cell lysates, and that their interaction is enhanced by ethanol in experimental models and in the livers of patients with alcoholic cirrhosis. Ethanol treatment in AML12 cells and in the NIAAA model increased MATα1 phosphorylation. Phosphoproteomics/MS revealed MATα1 is phosphorylated at Ser114 and mutation of Ser114 to alanine significantly reduced its interaction with PIN1. Increased PIN1‐MATα1 interaction reduced MATα1 mitochondrial targeting, as silencing PIN1 raised mitochondrial MATα1 content in AML12 and HepG2 cells, and attenuated ethanol‐induced triglyceride accumulation. Consistently, mitochondrial MATα1 content was preferentially reduced (by 60–80%) as compared to total MATα1 content (by 20–50%) in both in vitro and in vivo models and in livers from alcoholic hepatitis patients.ConclusionWe unveiled a novel mechanism of mitochondrial injury in ALD that involves PIN1. In ALD, increased MATα1 phosphorylation and interaction with PIN1 lead to impaired MATα1 mitochondrial targeting. Inhibiting PIN1‐MATα1 interaction may serve as a new therapeutic strategy in ALD.Support or Funding InformationThis work was supported by NIH grants R01AA026759 (SC Lu and J Van Eyk) and R24AA025017 (Sun Z).In ALD, increased MATα1 phosphorylation and interaction with PIN1 impaired MATα1 mitochondrial targeting.Figure 1