bluuebunny/crossref_metadata_2025 huggingface dataset:
Allosteric activation of the RNA and lipid binding capabilities of the picornaviral 3CD polyprotein
Authors: Boehr David D.
Published: April 2020

In the backdrop of a hostile cellular environment, viral genomes must encode processes to regulate the host cell’s machinery to allow for the efficient replication and packaging of new virus particles. These processes are encoded by relatively small genomes, and so viruses face a formidable information storage problem. Picornaviruses are positive‐strand RNA viruses, and first produce a large polyprotein that must then be cleaved to generate functional components. Proteolytic precursors and fully mature forms of viral proteins often have different functions. The picornavirus 3CD protein exemplifies this concept. The 3C protein is a protease and has RNA‐binding capabilities, and the 3D protein is an RNA‐dependent RNA polymerase (RdRp). The 3CD protein has unique protease and RNA‐binding abilities relative to 3C and is devoid of RdRp activity. How these functional changes in 3CD arise is poorly explained by its X‐ray crystal structure, which suggests that the 3C and 3D domains are merely tethered together by a linker (i.e. ‘beads‐on‐a‐string’ type model), and the domains do not interact. Computer simulations and small angle X‐ray scattering experiments are consistent with a dynamic 3CD conformational ensemble in which transient interactions between the domains may influence function. Our biophysical studies suggest an even simpler mechanism – extension of the C‐terminal tail of 3C by a few amino acid residues dramatically changes function. A network of interactions then transmits these structural/dynamic changes to the protease active site and the RNA‐binding site, which may also affect the ability of 3CD to interact with phosphoinositide‐containing viral replication membranes. Proteolytic processing of the C‐terminus is then the switch between 3CD and 3C/3D function. Such a simple mechanism may not require any additional domain‐domain interactions in the 3CD polyprotein to regulate domain function. In a broader context, we propose that the conformational dynamics of precursor and processed forms of viral proteins regulate the specific function of these molecules during the viral replication cycle to expand the functional proteome beyond the limited storage capacity available within small viral genomes.

Support or Funding Information

This work was funded through NIH R01 grants from NIAID: AI091985 and AI104878 (DDB) and AI053531 (CEC)

(A) X‐ray crystal structure of 3C (PDB 1L1N) highlighting the protease catalytic triad in black, and residues important for RNA and phosphoinositide lipid binding highlighted in green and orange, respectively. Binding of (B) phosphoinositide lipid and (C) RNA to the 3C constructs was measured using fluorescence polarization. The Dn designations indicate the number (n) of 3D residues added to the C‐terminus of 3C.

Figure 1
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 Information

Department 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

Introduction

Functional 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.

Results

Rate 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.

Conclusion

To 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 Information

AHA 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 embryo

This 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 Information

NY Spinal Cord Injury Research BoardMarine Biological Laboratory, Woods Hole, MA

Lamprey 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 Information

We 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

Introduction

Major 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.

Methods

7‐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.

Results

While 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).

Conclusions

We propose that insulin rather than glucose levels may be the driver of bladder enlargements in some models of diabetes and hyperinsulinemia.

Support or Funding Information

Deutsche 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/A

Correlation of bladder weight with glucose and insulin levels

Figure 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 Information

NONE

Example 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

Background

Methionine 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.

Methods

MATα1 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.

Results

PIN1 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.

Conclusion

We 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 Information

This 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
A novel ex‐vivo procedure for monitoring β‐hydroxybutyrate (βOHB) production from isolated whole livers
Authors: Singh Avinash , Chakraborty Saroj , Mell Blair , Wenceslau Camilla , McCarthy Cameron , Joe Bina
Published: April 2020

When glucose is limiting, such as during fasting, the liver synthesizes and secretes ketone bodies into circulation as an alternate fuel source. Previously, we found that the ketone body beta‐hydroxybutyrate (βOHB) is lowered by salt consumption in hypertensive rats. To identify hepatic‐specific mechanisms that regulate βOHB in vivo is difficult because extra‐hepatic factors impact liver function. Thus, we aimed to develop an ex vivo procedure to continually monitor hepatic βOHB. D‐glucose, but not L‐glucose, is a substrate for ATP generation. We exploited this difference to simulate non‐fasted (D‐glucose) and fasted (L‐glucose) conditions in vivo and hypothesized that livers incubated with L‐glucose will generate more βOHB compared to those incubated with D‐glucose. Portal veins of 27‐week‐old Wistar‐Kyoto rats (n=3/group) were perfused with either D‐glucose or L‐glucose in Krebs buffer. Whole livers were excised and incubated with the same buffers as perfusion. Aliquots were collected from 0–6hrs for quantitating βOHB. A statistically significant time effect was noted in both buffers, with βOHB continually increasing with time [D‐glucose Krebs (mmol/L), 0h: 0.03±0.00 vs. 6h: 0.82±0.02*; L‐glucose Krebs (mmol/L), 0h: 0.04±0.00 vs. 6h: 1.16±0.14*, both *p<0.05]. Confirming the sensitivity and validity of our preparation, there was a significant interaction effect, with livers incubated for 6 h with L‐glucose generating higher levels of βOHB than livers incubated with D‐glucose [D‐glucose: 0.82±0.02 vs. L‐glucose: 1.16±0.14*, *p<0.05). Our novel ex vivo liver preparation simulates fasting and non‐fasting conditions in vivo and can be applied to study liver‐specific regulation of βOHB independent of extra‐hepatic factors.

Support or Funding Information

American Heart Association (18POST34060003) and National Institutes of Health (R00GM118885 and R01HL143082).

Isolated, whole livers, incubated in Krebs buffer made with L‐glucose, have increased beta‐hydroxybutyrate synthesis compared to livers incubated in Krebs buffer made with D‐glucose. n=3. Two‐way ANOVA: *p<0.05.

Figure 1
The Role of the Actin‐Binding Protein Cortactin in the Onset and Development of Sepsis
Authors: Lartey Nathaniel Lartey , Ponce Alexander Garcia , Vargas Hilda , Shibayama Mineko , Schnoor Michael
Published: April 2020

Introduction

Sepsis is a devastating disease, which occurs as a result of an exacerbated immune response to systemic infection. Major hallmarks of sepsis include increased vascular permeability and leukocyte recruitment to secondary organs such as lung and kidney, often leading to organ dysfunction and, in severe cases, organ failure. Cortactin is an actin‐binding protein, expressed ubiquitously with the exception of neutrophils. Endothelial cortactin regulates vascular permeability and neutrophil recruitment by controlling activation of small GTPases. Therefore, endothelial cortactin could play an important role during sepsis pathogenesis by controlling neutrophil influx into affected organs such as lungs.

Aim/Objective

To elucidate the effects of cortactin deficiency on the onset and development of sepsis, which is a disease characterized by massive systemic neutrophil extravasation.

Experimental Strategy

Sepsis was induced by cecal ligation and puncture (CLP) in age‐matched male cortactin‐deficient (CTTN KO) and littermate wild‐type (WT) mice; and five day survival was monitored. Blood and lung tissue samples were collected 24 h after CLP for protein and gene expression analysis. Lung histology was analyzed after hematoxylin/eosin staining of paraffin‐crossections of lung tissue. Oxidative stress was assessed in lung cross‐sections using dihydroethidium. mRNA and protein levels of inflammatory cytokines were assessed by quantitative real‐time PCR (lung tissue) and bead‐based milliplex assay (peripheral blood) respectively. Neutrophil numbers were determined in blood and peritoneum by flow cytometry.

Results and Conclusions

Cortactin deficiency improved survival of mice subjected to CLP. Of note, lung tissue of septic KO animals showed better preserved architecture of the alveoli, and absence of oedema, hemorrhage and mucus deposition. Gene expression and secretion of the inflammatory mediators tumor necrosis factor‐α (TNFα) and interleukin‐1β in KO mice were reduced in comparison to septic WT mice. Moreover, we observed less oxidative stress in the lungs of septic KO mice in comparison to septic WT mice. Cortactin deficiency also protected against sepsis‐induced neutropenia. Additionally, in the absence of cortactin, less neutrophils were recruited to the peritoneum without compromising bacterial clearance. In summary, cortactin deficiency attenuates both local and systemic inflammatory responses during sepsis, protects against sepsis‐induced neutropenia and protects from lung tissue damage. Thus, cortactin could be an interesting target as novel treatment strategy for sepsis.

Cortactin deficiency protects against sepsis‐induced lung damage

Figure 1

Expression of inflammatory cytokines in the lung are reduced in the absence of cortactin

Figure 2
Effects of 1‐year Aerobic Exercise Training on Cerebral Blood Flow and Arterial Siffness in Amnestic Mild Cognitive Impairment
Authors: Tomoto Tsubasa , Pasha Evan , Sugawara Jun , Tarumi Takashi , Chiles Collin , Curtis Bryon , Cardim Danilo , Zhang Rong
Published: April 2020

Aerobic exercise training (AET) may improve cerebralperfusion and reduce the risk of dementia. Changes in central arterialstiffness and pulsatile cerebral blood flow (CBF) associated with exercise training may be one of the underlying pathophysiological mechanism. The purpose of this study was to investigate: (1) The effects of AET on central arterialstiffness and CBF, and (2) whether pulsatile CBF mediates the association between changes in arterial stiffness and cerebral perfusion in amnestic mildcognitive impairment (aMCI), a prodromal stage of Alzheimer disease. Seventy aMCI patients were randomized to 12 months of moderate‐intensity AET (n=31) or stretching program (n=39). CBF and carotid β‐stiffness index were assessed by ultrasonography and applanation tonometry. Cerebral perfusion was calculated by normalizing total CBF to brain tissue mass measured by magnetic resonance imaging. Pulsatile CBF was measured from the middle cerebralartery using transcranial Doppler. Cardiorespiratory fitness was assessed by peak oxygen uptake (VO2peak). VO2peak and cerebralperfusion increased and carotid β‐stiffness index decreased in the AET group, while remained unchanged in the stretching group. The change in VO2peak was significantly associated with the change in cerebral perfusion, carotidβ‐stiffness index, and pulsatile CBF (Fig.
1). The decrease in carotidβ‐stiffness was associated with increased in cerebral perfusion (Fig.
2). Mediation analysis showed that changes in pulsatile CBF mediates the association between carotidβ‐stiffness index and cerebral perfusion (Fig.
2). These results suggest that AET decreases arterial stiffness and increases cerebral perfusion, which is mediated by pulsatile CBF in aMCI patients.

Support or Funding Information

This work was funded by the National Institutes of Health ((R01AG033106, R01HL102457, P30AG012300)

Associations between the change (Δ) in relative peak oxygen uptake (VO2) and the change in cerebral perfusion, carotid β‐stiffness index and pulsatile cerebral blood flow (CBF).

Figure 1

Mediation analysis diagram of the effect of the change (Δ) in carotid β‐stiffness index on the change in cerebral perfusion through the change in pulsatile cerebral blood flow (CBF).

Figure 2
Epidural electrical stimulation and hemodynamic control after spinal cord injury
Authors: Soriano Jan Elaine , Saleem Saqib , Netoff Theoden I. , Squair Jordan W. , Samadani Uzma , Krassioukov Andrei V. , Darrow David , Phillips Aaron A.
Published: April 2020

Spinal cord injury impairs cerebral autoregulation (i.e., the buffering of changes in blood pressure in an effort to maintain cerebral blood flow constant) and baroreflex sensitivity (i.e., the appropriate heart rate responses to changes in blood pressure), due in part to the disruption of supraspinal sympathetic control of circuits below the level of injury. This impairment is almost certainly associated with the greater risk of cerebrovascular and heart disease observed in this population.

We have previously shown in people with spinal cord injury that epidural electrical stimulation of the spinal cord below the level of injury (eSTIM) activates disconnected sympathetic circuits. Furthermore, long‐term daily application of eSTIM can in some cases lead to plasticity that restores function, even when eSTIM is off. It is not clear if eSTIM activation of sympathetic circuits restores cerebral autoregulation and baroreflex function after spinal cord injury, or if functionally‐relevant plasticity occurs after long‐term application of eSTIM.

Here, we show that activating eSTIM immediately improves cerebral autoregulation and baroreflex sensitivity. These positive immediate effects were repeatable over several months. Long‐term daily eSTIM for at least 12 weeks did not affect cerebral autoregulation or baroreflex function when stimulation was off, indicating that functionally‐relevant plasticity did not occur. In conclusion, immediate application of eSTIM may be a viable therapy for improving hemodynamic control after spinal cord injury.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada, Canadian Institutes of Health Research, Libin Cardiovascular Institute of Alberta, Hotchkiss Brain Institute, Compute Canada.

Cerebral autoregulation was consistently improved with the activation of eSTIM as indicated by increased phase lag between blood pressure and cerebral blood flow. Cardiovagal baroreflex function was also improved as indicated by more rapid heart rate responses to changes in blood pressure. **indicates significant difference (p<0.005)

Figure 1
Maternal exercise programs skeletal muscle metabolic dysfunction in offspring mice challenged with high fat diet.
Authors: Son Jun Seok , Chen Yanting , Chae Song Ah , Chen Ke , Zhu Mei-Jun , Du Min
Published: April 2020

Exercise, including maternal exercise becomes increasingly less common. Though improper maternal nutrition and obesity are known to affect fetal muscle development and predisposes offspring to metabolic dysfunction, the effects of maternal exercise (ME) on fetal muscle development and offspring metabolic health are largely undefined.

PURPOSE

To investigate the effects of maternal exercise on mitochondrial biogenesis during fetal muscle development, and muscle function and metabolic health in offspring following high fat diet (HFD) challenge.

METHODS

Female C57BL/6J mice were randomized and assigned into a control (CON) or an exercise (EX) group (n = 6 per group) fed a normal chow. Exercise was performed every morning for 60 min during pregnancy. Then, the animals were mated, and the skeletal muscle samples were collected at embryonic day 18.5, weaning, and after 8 weeks of high fat diet (HFD; 60 kcal% from fat) challenge in both females and males. Statistical analysis were conducted using student’s t‐test and/or one/two way analysis of variance (ANOVA).

RESULTS

ME muscle fiber diameters and enhanced muscle endurance capacity in offspring (P < 0.05), which were positively associated with switching muscle fibers from type IIb/x to type I and IIa and mitochondrial biogenesis (P < 0.05), but negatively correlated with atrophy‐related Atrogin‐1 and Murf1 gene expression (P < 0.05). Mechanistically, ME enhanced DNA demethylation of Pgc‐1a promoter and its expression during fetal muscle development. Importantly, such changes in DNA methylation maintained in offspring muscle before and after HFD challenge, resulting in elevated Pgc‐1a mRNA expression and increased contents of PGC‐1a1 and 4 isoforms, which improved metabolic health.

CONCLUSIONS

This study identifies that ME enhances DNA demethylation of the Pgc‐1a promoter during fetal muscle development, which has programming effects on the exercise endurance capacity and metabolic health of muscle in offspring.

Support or Funding Information

This work was supported by National Institutes of Health Grants R01‐HD067449.

Maternal exercise (ME) enhances adaptation to endurance training of offspring mice (A,B) and the proportion of muscle type I fiber composition before and following HFD challenge (C). ME decreases muscle atrophy‐related genes following HFD challenge (D,E). Data are mean ± s.e.m. and each dot represents one litter.; two‐sided P values by unpaired Student’s t‐test (D,E) or two‐way repeated measures ANOVA followed by Tukey’s test (B).

Figure 1
Quantification of Mitochondrial Membrane Potential in the Isolated Rat Lung Using Rhodamine 6G
Authors: Audi Said H. , Cammarata Anthony , Clough Anne V. , Dash Ranjan K. , Jacobs Elizabeth R.
Published: April 2020

Mitochondrial membrane potential (ΔΨm) plays a key role in vital mitochondrial functions, and its dissipation is a hallmark of mitochondrial dysfunction. The objective of this study was to develop an experimental and computational approach for estimating ΔΨm in intact rat lungs using the lipophilic fluorescent cationic dye rhodamine 6G (R6G). Rat lungs were excised and connected to a ventilation‐perfusion system. The experimental protocol consisted of three single‐pass phases: loading, wash, and uncoupling, in which the lungs were perfused with R6G‐containing perfusate, fresh R6G‐free perfusate, or R6G‐free perfusate containing the mitochondrial uncoupler FCCP, respectively. This protocol was carried out with lung perfusate containing verapamil vehicle or verapamil, an inhibitor of the multi‐drug efflux pump P‐glycoprotein (Pgp). Results show that the addition of FCCP resulted in an increase in R6G venous effluent concentration (Figure
1), and that this increase was larger in the presence of verapamil than in its absence. A physiologically‐based pharmacokinetic (PBPK) model for the pulmonary disposition of R6G was developed and used for quantitative interpretation of the kinetic data, including estimating ΔΨm. The estimated value of ΔΨm (−139 ± 21 (SD) mV) was not significantly altered by inhibiting Pgp with verapamil, and is consistent with that estimated previously in cultured pulmonary endothelial cells. These results demonstrate the utility of the proposed approach for quantifying ΔΨm in intact functioning lungs. This approach has potential to provide quantitative assessment of the effect of injurious conditions on lung mitochondrial function, and to evaluate the impact of therapies that target mitochondria.

Support or Funding Information

Supported by NIH 2R15HL129209‐02 and VA Merit Review Award BX001681.

Solid symbols: R6G venous effluent concentrations during the loading phase, wash phase, and uncoupling phase using FCCP (67 μM). Values are mean ± SE (n = 7 for loading and wash phases, and n = 5 for uncoupling phase). Solid line is PBPK model fit to data.

Figure 1
Synthetic cadavers improved laboratory test grades in an undergraduate human anatomy course
Authors: Gregory Diane , Zwambag Derek , McFall Kristen , Andrews David , Richardson Nicolette
Published: April 2020

This study explored the use of an intriguing instrument which may provide a very similar, yet more sustainable, experience to human cadaver dissection in anatomy courses. The synthetic cadaver is structurally similar to a human cadaver but does not carry the same ethical and sustainability challenges. Students in an undergraduate human anatomy course completed two laboratory tests: Test 1 was completed prior to the introduction of two synthetic cadavers to the laboratory where the primary laboratory resources were plastic models. Test 2 was completed after the synthetic cadavers were introduced. Comparison of student lab test grades both pre and post synthetic cadaver introduction (“Experimental”) as well as to “Historical” cohorts of the course were used to assess student success. In addition, surveys comprised of 5‐point Likert items were used to assess student perceptions. Test 1 grades in the Experimental and Historical cohorts were compared to ensure that no academic differences existed between groups (p = 0.7653; z‐statistic = 0.2985). Students in the Experimental group performed better on Test 2 (median = 73.8% (95% CI: 72.0–75.0%) compared to the Historical group (70.1% (95% CI: 68.4%–70.1%), p < 0.0001). Students perceived laboratory resources to be necessary (95%), helpful (94%) and effective (87%), yet there was no change in these perceptions following the introduction of the synthetic cadavers. This investigation indicates that the synthetic cadavers were an effective learning tool in undergraduate human anatomy, and that student success was improved after using the synthetic cadavers, compared to when only plastic models were available.

Support or Funding Information

No funding was received for this work.

Probability density estimates for laboratory test grades in the Historical (light blue) and Experimental (dark blue) cohorts. Grey areas represent the 95% confidence interval for the median grade. There was no difference in median Test 1 grades; however, the median Test 2 grade in the Experimental cohort was significantly higher than in the Historical cohort.

Figure 1
Assessing and improving the performance of consensus docking strategies using the DockBox package
Authors: Preto Jordane , Gentile Francesco
Published: April 2020

Molecular docking is a well‐established computational method to predict how a ligand binds to a specific protein target as well as to assess the strength of the binding. Although docking programs are used worldwide for drug discovery, it is not always simple to identify which program or combination of programs provides the best results for a target of interest. Here we present DockBox, a computational package designed to facilitate the use and comparison of multiple docking and scoring programs at a time, and to combine them using different consensus strategies. A new consensus docking method called scoring‐based consensus docking (SBCD) is introduced, which significantly improves success rates in pose prediction as well as virtual‐screening (VS) hit rates compared to standard consensus docking. Several test cases are presented to illustrate the success of the SBCD method in docking and VS scenarios. SBCD requires the same computational cost as standard consensus methods in terms of number of required programs, computational power and time, making it a promising new approach for the screening of large chemical libraries.

Presentation of the DockBox package

Figure 1