bluuebunny/crossref_metadata_2025 huggingface dataset:
The splashback radius of optically selected clusters with Subaru HSC Second Public Data Release
Authors: Murata Ryoma , Sunayama Tomomi , Oguri Masamune , More Surhud , Nishizawa Atsushi J , Nishimichi Takahiro , Osato Ken
Published: April 2020

Recent constraints on the splashback radius around optically selected galaxy clusters from the redMaPPer cluster-finding algorithm in the literature have shown that the observed splashback radius is ${\sim}20\%$ smaller than that predicted by N-body simulations. We present analyses on the splashback features around ∼ 3000 optically selected galaxy clusters detected by the independent cluster-finding algorithm CAMIRA over a wide redshift range of 0.1 < zcl < 1.0 from the second public data release of the Hyper Suprime-Cam (HSC) Subaru Strategic Program covering ∼427 deg2 for the cluster catalog. We detect the splashback feature from the projected cross-correlation measurements between the clusters and photometric galaxies over the wide redshift range, including for high-redshift clusters at 0.7 < zcl < 1.0, thanks to deep HSC images. We find that constraints from red galaxy populations only are more precise than those without any color cut, leading to 1σ precisions of ${\sim}15\%$ at 0.4 < zcl < 0.7 and 0.7 < zcl < 1.0. These constraints at 0.4 < zcl < 0.7 and 0.7 < zcl < 1.0 are more consistent with the model predictions (≲1σ) than their $20\%$ smaller values as suggested by the previous studies with the redMaPPer (∼2σ). We also investigate selection effects of the optical cluster-finding algorithms on the observed splashback features by creating mock galaxy catalogs from a halo occupation distribution model, and find such effects to be sub-dominant for the CAMIRA cluster-finding algorithm. We also find that the redMaPPer-like cluster-finding algorithm induces a smaller inferred splashback radius in our mock catalog, especially at lower richness, which can well explain the smaller splashback radii in the literature. In contrast, these biases are significantly reduced when increasing its aperture size. This finding suggests that aperture sizes of optical cluster finders that are smaller than splashback feature scales can induce significant biases on the inferred location of a splashback radius.
Signature-dependent triaxiality for shape evolution from superdeformation in rapidly rotating 40Ca and 41Ca
Authors: Sakai Shinkuro , Yoshida Kenichi , Matsuo Masayuki
Published: April 2020

We investigate the possible occurrence of highly elongated shapes near the yrast line in $^{40}$Ca and $^{41}$Ca at high spins on the basis of the nuclear energy-density functional method. Both the superdeformed (SD) yrast configuration and the yrare configurations on top of the SD band are described by solving the cranked Skyme–Kohn–Sham equation in the three-dimensional coordinate space representation. It is suggested that some of the excited SD bands undergo band crossings and develop to hyperdeformation (HD) beyond $J \simeq 25 \hbar$ in $^{40}$Ca. We find that the change of triaxiality in response to rotation plays a decisive role in the shape evolution towards HD, and that this is governed by the signature quantum number of the last occupied orbital at low spins. This mechanism can be verified in an experimental observation of the positive-parity SD yrast signature-partner bands in $^{41}$Ca, one of which ($\alpha=+1/2$) undergoes crossings with the HD band, while the other ($\alpha=-1/2$) shows smooth evolution from collective rotation at low spins to non-collective rotation with an oblate shape at termination.
Coping with adversity: physical activity as a moderator in adaption to bereavement
Authors: Wicker Pamela , Orlowski Johannes
Published: April 2020

Background

The adaption of individuals to adverse life events such as bereavement which reduce their subjective well-being is referred to as the hedonic treadmill. This study examined whether previous physical activity moderates the negative impact of bereavement on subjective well-being and how quickly such adaption occurs.

Methods

The analysis uses data from 13 waves (1996–2015) of the German Socio-Economic Panel (n = 139 097). Subjective well-being is captured with respondents’ life satisfaction. Individual fixed effects regression models with three-wave lagged effects and interaction effects for bereavement and previous physical activity were estimated for females and males.

Results

The significant reduction in life satisfaction is partly offset for individuals who were physically active prior to the adversity, suggesting moderation effects of previous physical activity. These effects are larger in absolute value for females, but females also suffered more severely from bereavement. Males’ partial adaption through previous physical activity is closer to the pre-event level, while females adapt earlier. More frequent previous physical activity allows both genders to partially adapt more.

Conclusions

Previous physical activity helps individuals suffering bereavement to partially adapt faster to such adversity, suggesting that physical activity accelerates the hedonic treadmill, though at a different pace for each gender.
Memory, Attention, and Choice*
Authors: Bordalo Pedro , Gennaioli Nicola , Shleifer Andrei
Published: April 2020

Building on a textbook description of associative memory (Kahana 2012), we present a model of choice in which a choice option cues recall of similar past experiences. Memory shapes valuation and decisions in two ways. First, recalled experiences form a norm, which serves as an initial anchor for valuation. Second, salient quality and price surprises relative to the norm lead to large adjustments in valuation. The model unifies many well-documented choice puzzles, including the attribution and projection biases, inattention to hidden attributes, background contrast effects, and context-dependent willingness to pay. Unifying these puzzles on the basis of selective memory and attention to surprise yields multiple new predictions.
Clozapine Combination and Augmentation Strategies in Patients With Schizophrenia —Recommendations From an International Expert Survey Among the Treatment Response and Resistance in Psychosis (TRRIP) Working Group
Authors: Wagner Elias , Kane John M , Correll Christoph U , Howes Oliver , Siskind Dan , Honer William G , Lee Jimmy , Falkai Peter , Schneider-Axmann Thomas , Hasan Alkomiet ,   TRRIP Working Group
Published: April 2020

Background

Evidence for the management of inadequate clinical response to clozapine in treatment-resistant schizophrenia is sparse. Accordingly, an international initiative was undertaken with the aim of developing consensus recommendations for treatment strategies for clozapine-refractory patients with schizophrenia.

Methods

We conducted an online survey among members of the Treatment Response and Resistance in Psychosis (TRRIP) working group. An agreement threshold of ≥75% (responses “agree” + “strongly agree”) was set to define a first-round consensus. Questions achieving agreement or disagreement proportions of >50% in the first round, were re-presented to develop second-round final consensus recommendations.

Results

Forty-four (first round) and 49 (second round) of 63 TRRIP members participated. Expert recommendations at ≥75% agreement included raising clozapine plasma levels to ≥350 ng/ml for refractory positive, negative, and mixed symptoms. Where plasma level-guided dose escalation was ineffective for persistent positive symptoms, waiting for a delayed response was recommended. For clozapine-refractory positive symptoms, combination with a second antipsychotic (amisulpride and oral aripiprazole) and augmentation with ECT achieved consensus. For negative symptoms, waiting for a delayed response was recommended, and as an intervention for clozapine-refractory negative symptoms, clozapine augmentation with an antidepressant reached consensus. For clozapine-refractory suicidality, augmentation with antidepressants or mood-stabilizers, and ECT met consensus criteria. For clozapine-refractory aggression, augmentation with a mood-stabilizer or antipsychotic medication achieved consensus. Generally, cognitive-behavioral therapy and psychosocial interventions reached consensus.

Conclusions

Given the limited evidence from randomized trials of treatment strategies for clozapine-resistant schizophrenia (CRS), this consensus-based series of recommendations provides a framework for decision making to manage this challenging clinical situation.
AVATAR Therapy for Distressing Voices: A Comprehensive Account of Therapeutic Targets
Authors: Ward Thomas , Rus-Calafell Mar , Ramadhan Zeyana , Soumelidou Olga , Fornells-Ambrojo Miriam , Garety Philippa , Craig Tom K J
Published: April 2020

AVATAR therapy represents an effective new way of working with distressing voices based on face-to-face dialogue between the person and a digital representation (avatar) of their persecutory voice. To date, there has been no complete account of AVATAR therapy delivery. This article presents, for the first time, the full range of therapeutic targets along with information on acceptability and potential side effects. Interest in the approach is growing rapidly and this report acts as a necessary touchstone for future development.
Spatiotemporal control of gene expression boundaries using a feedforward loop
Authors: Reeves Gregory , Bandodkar Prasad , Al Asafen Hadel
Published: April 2020

A feed forward loop (FFL) is commonly observed in several biological networks. The FFL network motif has been mostly been studied with respect to variation of the input signal in time, with only a few studies of FFL activity in a spatially distributed system such as morphogen‐mediated tissue patterning. However, most morphogen gradients also evolve in time. We studied the spatiotemporal behavior of a coherent FFL in two contexts: (1) a generic, oscillating morphogen gradient and (2) the dorsal‐ventral patterning of the early Drosophila embryo by a gradient of the NF‐κB homolog Dorsal with its early target Twist. In both models, we found features in the dynamics of the intermediate node – phase difference and noise filtering – that were largely independent of the parameterization of the models, and thus were functions of the structure of the FFL itself. In the Dorsal gradient model, we also found that the dynamics of Dorsal require maternal pioneering factor Zelda for proper target gene expression.

Support or Funding Information

During this work, GTR and PB were partially supported by NIH grant (number R21HD092830), and GTR and HA were partially supported by NSF CAREER award (number CBET‐1254344).

A. Schematic figure depicting the generic morphogen gradient model. The inset shows the variation of the concentration of the primary transcription factor A in space and time. B. Schematic figure depicting the Dl/Twi/Zld model. The inset shows the variation of the concentration of the primary transcription factor Dl in space and time. The peaks in the Dl gradient amplitude in time correspond to nuclear cycles 11–14.

Figure 1
A Diet High in Saturated Fat Results in Greater Increases in Splenic Tregs in Female vs Male Sprague Dawley Rats
Authors: Ramirez Lindsey , Snyder Elizabeth , Sullivan Jennifer
Published: April 2020

Background

Diets high in saturated fats promote inflammation and are linked to the development of many cardiovascular diseases (CVD). Young females are typically protected from CVD vs males. However, epidemiological and basic science studies suggest that the cardiovascular protection in females is compromised by a high fat diet (HFD). Little is known regarding the impact of a HFD on T cells in males vs. females. Therefore, the current study tested the hypothesis that a HFD results in a more pro‐inflammatory T cell profile in females vs. males.

Methods

26‐day‐old male and female Sprague Dawley rats (n=3–5 per group) were randomized to a control (Ctrl; 7.2% calories from fat) or HFD (36% of calories from fat) for 10 weeks. 24 hour food consumption was measured every 3 weeks. Body weights were measured weekly. At the end of the 10 week treatment, body fat composition was measured via NMR. Rats were euthanized and spleen were isolated for flow cytometric analysis of Pan T cells (CD3+), CD4+ T cells (CD3+CD4+), and T regulatory cells (Tregs; CD3+CD4+FOXP3+).

Results

Throughout the treatment, rats on HFD consumed more kcal from fat vs Ctrls (P<0.05). There were no differences in body weight between Ctrl and HFD rats of the same sex (P>0.05 at each time point), although males were heavier than females at all time points (P<0.05), Table
. Following 10 weeks of treatment, HFD increased body fat percentage in both sexes vs Ctrl, (PDiet=0.01). Increases in body fat were similarly in both sexes (PSex=0.33; Pinteraction=0.37). At end of treatment, all groups had similar percentages of splenic CD3+ cells (Pdiet=0.27; Psex=0.14; Pinteraction=0.41). However, rats on a HFD had more CD4+ T cells vs Ctrl (PDiet=0.006). Moreover, females had more CD4+ T cells vs males (PSex=0.002; Pinteraction=0.09). HF females had the most splenic Tregs vs all groups (PDiet=0.28, PSex=0.09, PInteraction=0.007.

Discussion

This dietary model allows for investigation of the effects of a HFD on body composition and T cell count independent of obesity. Evidence in the literature suggests high anti‐inflammatory Tregs in female rats contributes to cardiovascular protection and decrease in renal Tregs compromises this protection. Thus, the increase in splenic Tregs in females on a HFD may show a compensatory increase to counteract the decreased renal Tregs observed in the literature. Future studies will test this hypothesis.

Data following 0 and 10 weeks of dietary treatment expressed as means ± SEM.

Assay

Weeks of treatment

Population

Ctrl F

HF‐F

Ctrl‐M

HF‐M

Body weight (g)

0

73.25 ± 0.48

70 ± 1.05

83.2 ± 2.13

80.2 ± 1.07

10

254 ± 8.57

242.4 ± 11.05

411 ± 11.79

410.8 ± 13.72

Body fat percentage

10

7.94 ± 0.49

11.35 ± 1.23

7.84 ± 1.69

10.18 ± 0.82

Splenic T cells

10

CD3

21.5 ± 1.55

20.96 ± 2.02

25.9 ± 2.3

22.28 ± 0.65

CD4

73.48 ± 1.38

77.36 ± 2.85

58.8 ± 2.91

72.23 ± 2.44

Tregs

6.05 ± 0.31

8.47 ± 0.79

7.55 ± 0.92

5.63 ± 0.32
Forbidden Protein Angles: Triosephosphate Isomerase as a Model for Studying Peripheral Residue Contribution to Catalysis.
Authors: Allen Patrick
Published: April 2020

Over the past 3.5 billion years of evolution, enzymes have adopted a seemingly infinite number of conformations to suit life on earth. However, torsional angles of proteins have settled into limited zones of energetically favorable dihedrals observed in Ramachandran plots. Areas outside said zones are believed to be forbidden to all amino acids, save glycine, due to steric hindrance. Triosephosphate Isomerase (TIM), a homodimer with a catalytic rate approaching the diffusion limit, contains an active site Lysine 13 (K13) with a dihedral within the forbidden zone (phi=69/psi=−139). Both the amino acid and the dihedral angle are conserved across all species of TIM and known crystal structures regardless of ligand. Only crystal structures of the engineered monomeric version (1MSS) show accepted β‐sheet dihedral values of phi=−135/psi=170 but experiments show a 1,000‐fold loss in activity. Based on these results, we hypothesized that adopting the forbidden torsion angle for K13 contributes to catalysis. Using a combination of computational and experimental approaches, four residues that interact with K13 (N11, M14, E97, and Q64) were mutated to alanine from single to quadruple mutants. In Silico Molecular Dynamic (MD) simulations of 5×200ns per mutant species were made using 2JK2 unliganded Human TIM as a starting structure. Heat Maps, containing 400,000 K13 dihedral values per run reveal full or partial loss of forbidden zone angles depending on mutant combination. Initial experimental results with E97A single mutant showed a three‐fold decrease in kcat with little change in the KM value suggesting a modest catalytic role for this residue. For this mutant the heatmaps revealed the K13 angle spends less than 30% of its time in the forbidden zone compared to over 50% occupation in the wildtype suggesting the rate decrease may be due to the mutation affecting the positioning of K13. In addition to ongoing In Vivo characterization of the remaining mutants, double mutant cycles will be used to evaluate their energetic connections with K13. As modern protein design relies on naturally occurring protein folds, understanding how to recreate forbidden angles may enhance catalytic rates in unnatural enzymes and achieve chemical reactions not yet envisioned by nature nor organic chemistry.

Support or Funding Information

We would like to thank California State University Long Beach and the Carl E. Riley Endowed STEM scholarship for their generous funding.

Heat map of where K13 dihedral occupies space on Ramachandran Plot over 200ns of MD simulation using Wild Type Human Triosephosphate Isomerase (RCSB:2JK2).

Figure 1
Role of Nuclear Receptor Condensates in Ligand Induced Co‐operative Gene Regulation
Authors: Nair Sreejith J , Yang Lu , Meluzzi Dario , Yang Feng , Oh Soohwan , Tan Yuliang , Gamliel Amir , Alshareedah Ibraheem , Wang Susan , Suter Tom , Friedman Meyer , Ma Qi , Jayani Ranveer , Zhang Jie , Aggarwal Aneel K , Banerjee Priya , Rosenfeld Michael G
Published: April 2020

Enhancers are transferable DNA elements that regulate target genes within the confines of Topologically Associated Domains (TADs) through Cohesin‐dependent looping. However, the potential regulatory functions and mechanisms of long‐distance, inter‐TAD enhancer associations remain largely unexplored. We report that 17β‐estradiol (E2) rapidly increases the spatial proximity of a fraction of E2‐responsive enhancers located in widely separated TADs. These enhancers are characterized by the recruitment of mega‐dalton sized multi transcription factor complex and high enhancer RNA (eRNA) induction. Optimal activation of these enhancer targets requires their co‐localization with phase‐separated interchromatin granules (ICGs). Chemical disruption of phase‐separated structures abolishes enhancer complex assembly and, specifically deactivates the most robust signal induced enhancers. Surprisingly, the physical property of the enhancer complex alters with the duration of hormone stimulation. In support of the long distance enhancer cooperativity model, the deletion of the most robust E2 induced enhancers resulted in a quantifiable decrease in the transcriptional activity of homotypic enhancers in the same chromosome. These data reveal previously unappreciated mechanisms for co‐ordinated gene regulation: (i) The biophysical properties of enhancer complex is critical for transcriptional output (ii) Phase separation mediated long‐distance enhancer cooperativity is required for optimal transcriptional activation by acute signaling programs. These findings reveal a new, generalizable model for the acute spatial gene regulation.

Support or Funding Information

Research support for S.J.N include postdoctoral fellowship from American Heart Association (14POST19860025), T32 Fellowship (5T32DK007044), and KO1 career grant (1K01DK121871). D.M. was supported by NIH‐NIDDK Grant 5F32DK112682. MGR is supported by Howard Hughes Medical Institute.

Model of E2‐induced changes in the chromatin architecture. E2 stimulation results in ERa dependent recruitment of MegaTrans complex, Condensin complex, and enhancer RNA as phase‐separated ribonucleoprotein condensates. Spatial enhancer association results from “megaloops” formation between the most robust enhancers (E.g. TFF1e1 and NRIP1e3). Maximal cooperative enhancer activation occurs with the co‐localization of the enhancers in the interchromatin granule (ICG).

Figure 1
Detection of transformed endothelial cells associated with survived cardiomyocytes in sub‐endocardial region of infarct myocardium
Authors: Song Xin , Xiao Ying , Chu Qing , Meng Xia , Kang Y. James
Published: April 2020

Previous studies have shown that cardiomyocytes in the sub‐endocardial region of infarct myocardium survive from ischemic injury. The present study was undertaken to explore a possible mechanism by which these cardiomyocytes survive in response to myocardial ischemia. Wild type mice (C57BL/6J, male, 8–10 weeks), or endothelial cell‐specific red fluorescent protein (RFP) expression transgenic mice (Tie2CRETdTomato) were subjected to permanent ligation of left anterior descending of coronary artery along with sham‐operated controls in which the same surgery procedure was applied with an exception of the artery ligation. Pimonidazole was injected intravenously to define the hypoxic region, and lectin was injected to trace blood infiltration before sacrifice in all of the mice. The hearts were harvested on day one after the surgery and cut into serial sections for detection of cardiomyocytes and endothelial cells. The staining of pimonidazole showed that most of the infarct myocardium was under hypoxic condition except the sub‐endocardial region in which survived cardiomyocytes were detected. The RFP positive endothelial cells in the sub‐endocardial region also survived but displayed a suppressed expression of PECAM‐1 and VEGFR2. Correspondently, lectin infiltration analysis suggested blood leakage in the sub‐endocardial region although there was no blood infiltration in other regions of the infarct area. The results suggest that blood infiltration through the transformed endothelial cells in the sub‐endocardial region of infarcted myocardium would provide a non‐hypoxic microenvironment, contributing to the survival of cardiomyocytes in this region.

Support or Funding Information

West China Hospital, Sichuan University

The changes of hypoxic microenvironment in the infarct myocardium region.

Hypoxic condition (partial pressure of O2 was less than 10 mmHg) was marked with hpi (pimonidazole) at 1st day after ischemic injury. The blood infiltrations were reflected by FITC‐conjugated WGA through tail vein at 1st, 4th, 7th day after LAD ligation. Hypoxic region stained by pimonidazole (red), and nuclear labeled by DAPI (blue). Abbreviations: DAPI, 4′,6‐diamidino‐2‐phenylindole; MI, myocardial infarction.

Figure 1

The change of blood supplement in the sub‐endocardial region.

The blood infiltrations were completely reflected by Lectin (FITC‐conjugated WGA) through tail vein at 1st day after LAD ligation. Cardiomyocytes stained by TNNI3 (red), Blood infiltration labeled by FITC‐conjugated WGA (green), and nuclear labeled by DAPI (blue). Abbreviations: WGA, wheat germ agglutinin; DAPI, 4′, 6‐diamidino‐2‐phenylindole; MI, myocardial infarction.

Figure 2
Translational pharmacology of TD139, an inhaled small molecule galectin‐3 (Gal‐3) inhibitor for the treatment of idiopathic pulmonary fibrosis (IPF)
Authors: Slack R. J. , Hirani N. , Gibbons M. A. , Simpson A.J. , Ford P. , Leffler H. , Nilsson U. J. , Sethi T. , Pedersen A. , Schambye H. , Maher T.M. , MacKinnon A. C.
Published: April 2020

Introduction

Gal‐3 is a pro‐fibrotic β‐galactoside‐binding lectin highly expressed in the lungs of IPF patients
1. TD139 is an inhaled, small molecule Gal‐3 inhibitor in development for IPF[2]. In this study the translational pharmacology of TD139 from pre‐clinical to clinical studies are presented.

Methods

TD139 was evaluated in pre‐clinical studies investigating Gal‐3 in vitro and in vivo in naïve and bleomycin‐treated mice (male C57/Bl6 mice)
1,
2. A randomized, double‐blind, multi‐centre, placebo‐controlled, phase IIa study was completed to assess safety, tolerability, and PK/PD of TD139 in 24 patients with IPF. Inhaled TD139 was delivered at 0.3 mg, 3 mg or 10 mg QD to IPF patients (8 patients/cohort, 5:3 ratio (active:placebo)) for 14 days. Patients underwent bronchoalveolar lavage (BAL) prior to dosing and after 14 days. TD139 drug concentration was measured in the BAL fluid, BAL macrophages and plasma. Gal‐3 expression on BAL macrophages was measured by flow cytometry and systemic biomarkers of Gal‐3 inhibition and fibrosis measured in plasma.

Results

TD139 demonstrated high affinity (KD = 2.1 ± 0.1 nM (mean ± SD, n=4)) in vitro and reduced Gal‐3 expression ex vivo on IPF macrophages (IC50 = 361 ± 108 nM (mean ± SD, n=3 patients)). TD139 in vivo attenuated mechanistic PD (BAL Gal‐3) and fibrotic endpoints (total lung collagen/histology) in bleomycin‐treated mice with duration driven by lung retention. Clinically, inhaled TD139 was well tolerated at all doses with concentrations in BAL macrophages >567‐fold higher than systemic exposure. Gal‐3 expression on BAL macrophages was significantly lower in the 3 mg (% change −52.52%, 95% CI −85.43% to −4.74%, P=0.023) and 10 mg (% change −78.60%, 95% CI −110.48% to −29.78%, P<0.0001) dose groups compared to placebo, with a concentration‐dependent inhibition. Reduced Gal‐3 expression on BAL macrophages was associated with lower plasma biomarkers relevant to IPF pathobiology (PAI‐1, YKL‐40 and PDGF‐BB).

Conclusion

TD139 is safe and well tolerated in man and demonstrates low systemic exposure coupled with high lung concentrations resulting in suppression of Gal‐3 on BAL macrophages and decreased plasma biomarkers associated with IPF progression. Pre‐clinical in vitro and in vivo studies have shown to be clinically translatable. This study supported the progression of TD139 into a phase IIb IPF study
3.

Support or Funding Information

All studies were funded by Galecto Biotech AB.
Sympathetic β‐Adrenergic Stimulation Evokes Less Positive Inotropic Response But More Ventricular Arrhythmias in Heart Failure
Authors: Greco Lisa V. , Li Ying , Zhang Youhua
Published: April 2020

Introduction

It is well‐known that heart failure (HF) results in impaired sympathetic autonomic signaling in the heart, known as “β‐adrenergic receptor downregulation or desensitization”. This results in reduced positive inotropic effect under β‐adrenergic stimulation in HF. Our recent data indicate that HF significantly alters sympathetic stimulation induced atrial fibrillation (AF) inducibility. In contrast to normal hearts, failing hears are more vulnerable to sympathetic stimulation induced AF. This study is to investigate whether ventricular arrhythmia inducibility is altered in HF by sympathetic β‐adrenergic stimulation.

Methods

HF was induced in 7 rats 2 months after myocardial infarction surgery with left coronary artery ligation, and 6 animals with sham surgery served as normal controls. Left ventricular pressure was recorded and the adrenergic β1‐agonist dobutamine (25μg/kg, iv) was administered to evoke positive inotropic effect. Ventricular arrhythmia inducibility test was performed with caffeine plus dobutamine challenge (caffeine 50mg/kg, ip, followed by dobutamine 25μg/kg, ip) and surface ECG as well as intracardiac right atrial electrocardiograms were recorded.

Results

Dobutamine induced positive inotropic effects were significantly less in HF rats than in sham‐controls (+dp/dt increased 49±13% in control versus 14±6% in HF, P<0.001). Caffeine and dobutamine challenge induced severe ventricular tachyarrhythmias (frequent premature ventricular contractions and ventricular tachycardia, figure) in 6/7 HF rats while no ventricular arrhythmia was induced in the controls (p<0.01).

Conclusion

Compared with normal hearts, failing hearts are much more vulnerable to develop severe ventricular tachyarrhythmia during sympathetic β‐adrenergic stimulation with dobutamine and caffeine challenge, despite β‐adrenergic receptor downregulation in HF, as reflected by the less positive inotropic effects induced by β‐adrenergic stimulation.

Example of induced premature ventricular contractions (PVC) and ventricular tachycardia (VT)

Figure 1
Myocardial Ischemia‐Reperfusion Injury: Does Disruption of Mitochondrial Cristae Integrity Play a Requisite Mechanistic Role?
Authors: Kulek Andrew , Raghunayakula Sarita , Anzell Anthony , Undyala Vishnu , Sanderson Thomas , Przyklenk Karin
Published: April 2020

Lethal myocardial ischemia‐reperfusion (IR) injury (i.e., cardiomyocyte death resulting in ‘heart attack’ or myocardial infarction, MI) is the leading global cause of death and disability. Despite improvements in the clinical management of MI, the molecular mechanisms responsible for lethal IR injury are incompletely understood. Data from our group and others have revealed that cardiomyocytes subjected to IR display a loss in integrity of optic atrophy protein (OPA1), the master‐regulator of fusion at the inner mitochondrial membrane and putative gate‐keeper of cristae junction integrity. Our goal in the current study was to establish whether disruption of OPA1 integrity plays a causal role in determining cardiomyocyte fate. To test this concept, HL‐1 cardiomyocytes were subjected to 2.5 hours of simulated ischemia + reperfusion. In
Protocol 1, the cleavage and subsequent release of OPA1 from mitochondria into the cytosol was assessed by immunoblotting at 30 min post‐R. As expected, IR was associated with degradation of OPA1 (in particular, the long (L) forms of the protein critical for inner mitochondrial membrane architecture), and a >15‐fold increase in cytosolic expression of the lower molecular weight short (S) forms (data not shown). In
Protocol 2, to demonstrate cause‐and‐effect, HL‐1 cells were transfected with either siRNA targeting OPA1 or scrambled peptide before undergoing 2.5 hours I + 24 hours R or a time‐matched normoxic period. Additional non‐transfected cells served as controls. Using 50 nM siRNA, near‐total knockdown (KD) of OPA1 (most notably, the critical L‐forms) was achieved (Figure
). In the normoxic cohorts, cardiomyocyte viability (quantified using the MTT assay) was modestly but significantly reduced in siRNA‐transfected cells (Figure
– left: *p<0.05 versus normoxic non‐transfected cells). However, contrary to the premise of our study, OPA1 knockdown had no effect on viability in cells subjected to IR: i.e., did not exacerbate IR‐induced cardiomyocyte death (Figure
– right). These data suggest that maintenance of OPA1 integrity does not have a requisite, causal role in mediating lethal myocardial ischemia‐reperfusion injury.

Support or Funding Information

Detroit Cardiovascular Training Grant T32 (NIH 2T32HL120822) through the National Heart, Lung, and Blood Institute ‐ Wayne State University

OPA1 Knockdown with siRNA

Figure 1

HL‐1 Cell viability with OPA1 Knockdown

Figure 2
Generation of the Configurational Ensemble of an Intrinsically Disordered Protein
Authors: Petridis Loukas
Published: April 2020

A major challenge in biology is characterizing the structural flexibility of intrinsically disordered proteins (IDPs). Ensemble‐averaged experimental data do not provide the underlying protein structures. Here, we performed independently small‐angle neutron and X‐ray scattering experiments and unbiased molecular dynamics simulations to probe the solution structure of an IDP. We report that enhancing the sampling of the simulations can generate an ensemble of IDP structures in quantitative agreement with scattering and NMR, without the need for biasing the simulation or reweighting the results. The demonstration of established simulation technology that produces accurate physical models of flexible biosystems may pave the way to relating conformational flexibility to biological function.

Support or Funding Information

This work is supported by the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory, and by project ERKP300 funded by the Office of Biological & Environmental Research in the Department of Energy Office of Science.

Configurational ensemble of c‐Src kinase

Figure 1
Development and Characterization of a Novel Small Animal Model of Heart Failure with Preserved Ejection Fraction
Authors: LaPenna Kyle B , Hidalgo Hunter A , Gao Juan , Haydel Amelia , Kapusta Daniel , Goodchild Traci , Lefer David J
Published: April 2020

Heart Failure with Preserved Ejection Fraction (HFpEF) is a complex heterogeneous disease that currently affects approximately 3 million in the US and accounts for 60% of all heart failure hospitalizations. HFpEF is associated with significant morbidity, mortality and healthcare expenditures. At present, there are no FDA approved therapies for reducing mortality and hospitalization in HFpEF patients. The lack of approved therapies is due in part to a lack of preclinical models that faithfully mimic the clinical presentation of HFpEF.

The obese ZSF‐1 rat is genetically predisposed to the development of hypertension, obesity, dyslipidemia, and diabetes, all of which contribute to the pathogenesis of HFpEF. We investigated the suitability of the obese ZSF‐1 rat as a model of HFpEF. Both obese and lean control ZSF‐1 rats (n = 4–6 per group) were utilized for these studies. The rats were studied from 16 weeks to 24 weeks of age. We measured several clinical parameters including cardiac structure and function (2‐D echocardiography), left ventricular (LV) pressures (LV Millar catheter), cardiac fibrosis, conscious blood pressure and heart rate (DSI radiotelemeters), vascular function, exercise capacity (rodent treadmill), and serum lipid and triglyceride levels (standard blood ELISA assays).

Obese ZSF‐1 rats exhibited a significant increase in body weight and systemic blood pressure combined with hyperglycemia and insulin insensitivity. Echocardiographic evaluation of the left ventricle (LV) revealed highly significant increases in both the E/A and E/E′ ratios. LV end‐diastolic pressure (LVEDP) and LV relaxation (Tau) were decreased in the obese ZSF‐1 rats. Histological evaluation of the hearts revealed significant increases in cardiac fibrosis and cardiac myocyte injury in the obese vs. lean ZSF‐1. Furthermore, vascular reactivity and circulating nitric oxide levels were reduced in the obese ZSF‐1 rats. Exercise capacity testing revealed that exercise capacity was very significantly reduced in the ZSF‐1 obese rat throughout the 8 week experimental protocol. All these findings are consistent with the HFpEF phenotype observed in human HFpEF patients.

This preclinical model of HFpEF, will provide novel insights into the pathobiology of HFpEF and provide a critically important platform for testing HFpEF therapeutics. Our future studies will be aimed at the identification of novel therapeutic interventions to treat HFpEF.

Support or Funding Information

National Institutes of General Medical Sciences (NIGMS) COBRE grant no. P30GM106392 to Dr. Kapusta

Figure 1
Protein Kinase A (PKA) Catalytic Subunits a and b Have Non‐Redundant Functions in Collecting Duct Cells
Authors: Salhadar Karim , Raghuram V. , Yang Chin-Rang , Limbutara Kavee , Knepper Mark
Published: April 2020

Vasopressin regulates osmotic water transport in the renal collecting duct by PKA‐mediated control of the water channel aquaporin‐2 (AQP2). Collecting duct principal cells express two seemingly redundant PKA catalytic subunits, PKA catalytic α (PKA‐Cα [Gene symbol: Prkaca]) and PKA catalytic β (PKA‐Cβ [Gene symbol: Prkacb]). To identify the relative roles of these two proteins, we carried out deep phosphoproteomic analysis in cultured mpkCCD cells in which either PKA‐Cα (n=3) or PKA‐Cβ (n=3) was deleted using CRISPR‐Cas9‐based genome editing (generated in PMID 28973931). Cells were grown on permeable supports in the presence of the vasopressin analog dDAVP (0.1nM). Controls were cells carried through the genome editing procedure, but without deletion of PKA. Quantification used TMT mass tags in an Orbitrap Fusion Lumos ETD mass spectrometer. Preliminary measures of total protein amounts showed that PKA‐Cα deletion resulted in a near disappearance of AQP2 protein, while PKA‐Cβ deletion was associated with an
increase in AQP2. A total of 4635 phosphopeptides were quantified (Figure
1). The red data points represent phosphopeptides that correspond to previously identified PKA target sites. Stringent statistical criteria (Pmod < 0.01 and |log2(KO/control)| > 0.503) revealed that 67 phosphopeptides were significantly altered with PKA‐Cα deletion, while 21 phosphopeptides were significantly altered with PKA‐Cβ deletion. Only four sites were changed in both. We used Gene Ontology (GO‐CC) Cellular Component terms to identify sets of target phosphoproteins localized in different regions of the cell. The target proteins identified in PKA‐Cα‐null cells were largely associated with cell membranes and membrane vesicles, while target proteins in the PKA‐Cβ‐null cells were largely associated with the actin cytoskeleton and cell junctions. In summary, PKA‐Cα and PKA‐Cβ appear to serve substantially different functions in renal collecting duct cells. The differences in the phosphorylation targets of PKA‐Cα and PKA‐Cβ suggest differences in the cellular localization of the two PKA subunits. These findings in addition to the dramatically different effect of PKA‐Cα and PKA‐Cβ deletion on AQP2 protein abundance support the conclusion that PKA‐Cα and PKA‐Cβ have non‐redundant functions in collecting duct cells.

Support or Funding Information

K.S. was supported by the Biomedical Engineering Summer Internship Program at NIH and carried out all of the mass spectrometry experiments. Work was supported by NHLBI Project ZIA‐HL001285.

Volcano plots showing effect of PKA‐Cα deletion (left) and PKA‐Cβ deletion (right) on phosphoproteome of mouse mpkCCD cells. Three biological replicates for each.

Figure 1
Shikimate Kinase Expression; Towards Screening of Potential Antibacterials from Marine Sponges
Authors: Ogrodniczuk Marcin , Motilall Anand , Fuanta René
Published: April 2020

Tuberculosis is one of the world’s leading cause of mortality from a single bacterial pathogen, with over 10 million reported cases each year. There is an alarming increase in the prevalence of drug‐resistant strains, thus the need for the discovery of novel anti‐tubercular agents. The shikimate pathway is a seven‐step metabolic route that produces aromatic amino acids and other cellular metabolites. It has no mammalian counterpart, making any of the enzymes in this pathway suitable targets for screening of potential anti‐tubercular agents. Mycobacterium tuberculosis shikimate kinase (MtSK) catalyzes the 5th step of this pathway, converting shikimate to shikimate‐3‐phosphate using ATP as a co‐substrate. The overall goal of this project is to express and characterize MtSK and screen for potential anti‐tubercular agents. Transformation of XL‐1 blue competent cells was performed using a pET 21b plasmid with aroK gene inserted at the multiple cloning site. Plasmids were cloned and purified and used to transform BL 21 DE3 competent cells for subsequent protein expression. Small scale expression showed the presence of a band of increasing prominence around 20 kDa. This suggests MtSK was successfully expressed. Expression analysis for larger scale supported data from small scale. Purification, characterization, and MtSK kinetic parameters would be determined prior to enzyme inhibition studies using inhibitors like avarone (below), a marine sponge sesquiterpene quinone and derivatives thereof.

Support or Funding Information

East Stroudsburg University of Pennsylvania

Avarone

Figure 1
Assessment of Quadriceps Contraction Using a Novel Surface Mechanomyography Sensor During a Neuromuscular Control Screening Task
Authors: Linderman Shannon , Aspenleiter Ryan , Stein Hannah , Berkson Eric
Published: April 2020

PURPOSE

Neuromuscular control is an important factor for injury incidence and post‐surgical rehabilitation. Surface Mechanomyography (sMMG) sensors are novel wearable devices that are applied over a muscle group to measure the physical output of muscle deformation resulting from a muscle contraction. Electromyography (EMG) is the clinical standard for assessing the electrical signal identifying muscle activation. However, well‐established data collection and analysis challenges limit the utility of EMG for wide‐spread neuromuscular control screening in a clinic environment. The purpose of this study was to investigate the ability of sMMG sensors to detect timing patterns of muscle contraction and compare time events to those collected through traditional clinical EMG during a bilateral squat task.

METHODS

Eleven healthy, active individuals (mean age= 30.0 ± 10.77 y, 7 males, 4 females) underwent a neuromuscular control assessment with EMG and sMMG sensors simultaneously applied to the right quadriceps. Subjects performed a series of 3 bilateral deep squats. EMG data processed with a low‐pass 6th order Butterworth filter and a TKEO function, and raw sMMG data were used for timing analyses. Statistical analyses included paired T‐test assessments between measurement modalities.

RESULTS

There was no significant difference in the timing of total duration of quadriceps contraction between EMG (mean= 2.526 ± 0.553 s) and sMMG (mean= 2.527 ± 0.539 s), p= 0.985. The duration of quadriceps contraction during the descent phase of the squat (eccentric contraction, p= 0.773) and the ascent phase (concentric contraction, p= 0.298) did not differ significantly between modalities.

CONCLUSIONS

Results are consistent with physiologic expectations that myoelectrical activity (measured by EMG) and the physical muscle deformation of muscles (measured by sMMG) occur in extremely rapid succession. Successful sMMG detection of quadriceps contraction is supported by similarity to EMG time signatures. Findings also suggest the ability of sMMG sensors to detect timing of activation for different types of muscle contraction during a functional exercise without the need for complex signal processing (Figure
1). The sMMG sensor may be helpful for assessing quadriceps muscle performance and timing as part of quick, in‐the‐clinic neuromuscular control screenings for injury prevention, rehabilitation, and exercise training.

Example of data analysis steps for comparison between electromyography (EMG) and surface mechanography (sMMG) timing during a bilateral squat task

Figure 1
Postnatal administration of leptin antagonist mitigates susceptibility to obesity under high‐fat diet in αMUPA male mice
Authors: Gutman Roee
Published: April 2020

Perturbations in postnatal leptin signaling have been associated with altered susceptibility to diet‐induced obesity (DIO), under high‐fat‐diet (HFD), albeit with contradicting evidence. Previous studies have shown that Alpha murine urokinase‐type plasminogen activator (αMUPA) mice have a higher and longer postnatal leptin surge compared with their wild types (WT), as well as lower body weight and food intake under regular diet (RD). Here we explore αMUPA’s propensity to DIO and the effect of attenuating postnatal leptin signaling, using leptin antagonist, on energy homeostasis under both RD and HFD. Four‐day‐old αMUPA pups were treated on alternate days until P‐18 with either vehicle or leptin antagonist (LA; 10 or 20 mg d−1 kg−1) and weaned into RD or HFD. Compared with RD‐fed αMUPA males, HFD‐fed αMUPA males showed higher energy intake, even when corrected for bodyweight difference, and became hyperinsulinemic and obese. Additionally, HFD‐fed αMUPA males gained bodyweight at a higher rate than their WTs due mainly to strain‐differences in energy expenditure. LA administration did not affect strain differences under RD, but attenuated αMUPA’s hyperinsulinemia and DIO under HFD, most likely by mediating energy expenditure. Together with our previous findings, these results suggest that αMUPA’s leptin surge underlies its higher susceptibility to obesity under HFD, highlighting the role of leptin‐related developmental processes in inducing obesity under post weaning obesogenic environment, at least in αMUPA males. This study, therefore, supports the use of αMUPA mice for elucidating developmental mechanisms of obesity and the efficacy of early‐life manipulations via leptin surge axis in attenuating DIO.

Support or Funding Information

From the Union’s Seventh Framework Program FP7‐REGPOT‐2012‐2013‐1, Agreement no. 316157

The effect of high‐fat diet (HFD) compared with regular diet (RD) on energy homeostasis of saline‐treated αMUPA and FVB/N wild‐type male pups. (A) Body weight; (B) body weight and composition at nine months of age; (C) energy balance. Abbreviations: FFM, fat‐free mass. N, 6–7 mice per treatment. Non‐matching lowercase letters indicate statistically significant differences among groups. *, statistically significant difference from zero, P<0.05. Results are presented as mean ± SEM.

Figure 1

The effect of leptin antagonist (LA) compared with saline on αMUPA under regular (RD) and high‐fat (HFD) diets, also compared HFD‐fed saline‐treated WT. Bodyweight (A) and composition at 9 mo of age (B); (C) Energy balance. Abbreviations: FFM, fat‐free mass; LA‐10, 10 mg·day‐1·kg‐1 of LA injections; LA‐20, 20 mg·day‐1·kg‐1 of LA injections. d, Day; Δ, change. Nonmatching letters indicate differences among groups. * and # difference from zero, P < 0.05 and P < 0.01.

Figure 2
Codon Optimization of Gene Editing CRISPR‐SaCas9 Plasmid Increases Protein Expression in Human Liver Cells to Boost in vivo Therapeutic Knockout Application Efficiency
Authors: Groshong Tyler L , Madejski Irene R , Sundaresan Avirath , Gupta Ishir G , Holmes Grace D , De Luke
Published: April 2020

The CRISPR‐Cas9 gene editing technology shows great promise in revolutionizing the treatment of genetic disease; however, the current focus on treatment by ex vivo delivery is limited to blood diseases. In vivo treatment can substantially extend the clinical reach of the technology, but is limited by a challenging delivery process: the large size of the Cas9 gene (~4500bp) inhibits delivery by the small viral vectors and lipid nanoparticles used. We propose that a codon‐optimized saCas9 plasmid will lead to significantly higher protein expression than the presently used genes; this can compensate for the low delivery efficiency and has great potential to improve the viability of in vivo CRISPR‐Cas9 therapies. Here we present Western Blot fluorescence data showing that optimization leads to increased Cas9 production and subsequently increased CRISPR‐Cas9 editing efficiency.

A commonly used parent saCas9 sequence was selected from Addgene’s public plasmid database. A novel, codon‐optimized gene sequence was engineered from the parent using GenScript’s OptimumGeneTM algorithm. Both the optimized gene and parent gene were transfected into human HepG2 liver cells, and Western Blot/ELISA protein quantification of the optimized plasmid compared with three sample plasmids demonstrated that the codon‐optimized gene expressed the saCas9 protein at ~3.5× the rate of the commercial sequence. The same set of optimized and commercial plasmids were then transfected into GFP‐stable HepG2 cells in parallel with GFP‐specific sgRNA; the cells transfected with optimized saCas9 demonstrated a significantly higher GFP knockout rate than cells transfected with commercially available control plasmids, as measured through GFP detection stay and Western Blot on GFP proteins. The augmented Cas9 protein expression supported by the novel saCas9 sequence can better enable in vivo CRISPR‐Cas9 treatments.

Support or Funding Information

This work was supported by The Nueva School.

Comparison of Commercial and Optimized SaCas9 Expression

Figure 1

SaCas9 Protein Standard Curve

Figure 2