bluuebunny/crossref_metadata_2025 huggingface dataset:
P144 Validation of the Musculoskeletal Health Questionnaire (MSK-HQ) in patients with musculoskeletal pain in primary care
Authors: Scott Ian C , McCray Gareth , Lancaster Gillian , Foster Nadine E , Hill Jonathan C
Published: April 2020

Background

The Musculoskeletal Health Questionnaire (MSK-HQ) is a recently developed generic patient-reported outcome measure (PROM), evaluating impacts of musculoskeletal (MSK) conditions on patients’ health. Its scores range 0-56; higher scores indicate better health. Its performance (a) in patients with MSK-pain in primary care, and (b) compared to MSK pain-site reference PROMs, is uncertain. We addressed these uncertainties through secondary analysis of a previous primary-care based study, enrolling 524 patients with shoulder, neck, lower back, knee, or multi-site MSK pain managed by GPs, from 8 West Midlands practices.

Methods

The study captured the following self-report questionnaire data at 0 and 6 months: MSK-HQ, EQ-5D-5L, Roland-Morris Disability Questionnaire (back pain), Neck Disability Index (neck pain), Shoulder Pain and Disability Index (shoulder pain), Knee Injury and Outcome Score (knee pain), Short-Form-12 (multisite pain). At 6 months, patients self-rated their global change in MSK pain, from -5 (“very much worse”) to + 5 (“completely recovered”). Receiver operating characteristic curves evaluated the ability of 6-month changes in each PROM to discriminate between patients improving/not improving on global change scores. Minimal Clinically Important Differences (MCID) were calculated (cut-off optimising discriminatory sensitivity/specificity).

Results

Most (60%) reported pain improvements, with mean baseline MSK-HQ scores rising from 29.4 to 37.4 over 6-months. The MSK-HQ had a moderate ability to discriminate between patients improving vs. no change/worsening in MSK pain (area under the curve [AUC] 0.81; 95% CI 0.78-0.85). Its discriminative ability was better than the EQ-5D-3L (AUC 0.68; 95% CI 0.62-0.73) and at least as good as site-specific PROMs (Table). MCID for the 6-month change in MSK-HQ was 5.5 across all patients. Minor variation (<2.0 units) was observed in MSK-HQ MCID across 4/5 pain sites. The MSK-HQ had strong correlations with all PROMs except SF-12 physical/mental component summary scores.

Conclusion

In patients consulting in primary care with MSK pain, the MSK-HQ appears at least as good as existing pain-site specific PROMs at identifying patients self-reporting pain improvements, and superior to the EQ-5D-5L. Our results support the use of the MSK-HQ in this setting.

Disclosures

I.C. Scott None. G. McCray None. G. Lancaster None. N.E. Foster None. J.C. Hill None.
P275 The extent and characteristics of diagnostic delay in axSpA: a systematic review
Authors: Hay Charles A , Ryan Sarah , Packham Jon , Mallen Christian D , Prior James A
Published: April 2020

Background

Axial spondyloarthritis (axSpA) is an inflammatory arthritis predominantly affecting the spine. It is characterised by chronic back pain, stiffness and fatigue. As axSpA progresses it can cause disability, reduction in quality-of-life, depression and impacts on work. Prompt diagnosis is important, but symptoms associated with axSpA are common in the general population resulting in diagnostic delay. The reported diagnostic delay for axSpA varies in the literature, ranging from five to fifteen years. The aim of this review was to ascertain the extent of axSpA diagnostic delay and report on axSpA characteristics associated with diagnostic delay.

Methods

A systematic review was conducted to identify articles reporting diagnostic delay in axSpA. Inclusion criteria were studies including adult axSpA populations, cohort, cross-sectional or case-control design and reporting a median time-period of delay from axSpA symptom onset to final axSpA diagnosis (studies reporting mean delay were excluded due to skewness of data). Data was also extracted which related to delay as a result of specific axSpA characteristics. Narrative synthesis was used to report our findings.

Results

9,848 articles were initially identified. After title, abstract and full-text review, 15 articles reported median diagnostic delay. Of these, 2 were from the UK, 2 from Germany, and 1 each from India, Norway, France, USA, Iran, Turkey, China and Italy. Combined, the total number of participants across eligible papers was 10,661. Though median delay ranged from ≤1 to 8 years, the majority (60%) of articles reported a median delay of between 2-5 years. Regarding the role of specific axSpA characteristics on delay, the most noted causes for delay were lack of extra-articular axSpA symptoms (5.9 vs. 8.7 years of delay for patients with and without extra-articular symptoms respectively) and misdiagnosis. Common misdiagnoses were lumbar disc herniation, rheumatoid arthritis and mechanical back pain (MBP), with the latter being significantly associated with delayed diagnosis (OR 2.83(95% CI 1.16-6.87)). Finally, delay is far from uniform, with a UK study reporting that 30.3% of patients are diagnosed within 2 years, 21% in 3-5 years, 19.9% in 6-10 years, 19.2% after 11-20 years and 9.6% after 20 years.

Conclusion

Though diagnostic delay of axSpA is considerable, affecting long term outcomes in a large number of patients, it is not as long as previously reported by using mean delay, which is more affected by bias (long term delays in small numbers of individuals) than median delay. Further research into the specific barriers and facilitators of delay is required to help reduce this delay in the future.

Disclosures

C.A. Hay None. S. Ryan None. J. Packham None. C.D. Mallen None. J.A. Prior None.
“Biting Our tongues”: Policy Legacies and Memories in the Making of the Syrian Refugee Response in Jordan
Authors: Lenner Katharina
Published: April 2020

This article analyses the significance of policy legacies and policy memories for refugee policy in conflict-neighbouring countries, where most of the world’s displaced live. Drawing on insights from critical policy analysis, it views refugee policy as co-produced by national and international agencies on the basis of previous dynamics that are already the product of an intense history of interaction and translation. This approach is illustrated by analysing two different aspects of refugee policy in Jordan: the process of counting Syrians in the country and the partial integration of Syrians into the formal labour market. Both examples reveal an overarching legacy of accommodation that ties international and host government actors together. Despite sometimes differing over preferred outcomes, the main goals for the various actors involved have been to strike compromises, safeguard organisational interests, and create outward policy success. In order to meet these goals, the agencies involved have learned to tolerate unresolved ambiguities and disregard other inconvenient legacies and memories that would only complicate policy negotiations. Acknowledging this intertwinement of agencies, technologies, and rationales of government is essential for rethinking policy change and responsibility in contexts of mass displacement.
The gray matter volume of the temporoparietal junction varies across cultures: a moderating role of the dopamine D4 receptor gene (DRD4)
Authors: Kitayama Shinobu , Yu Qinggang , King Anthony P , Yoon Carolyn , Liberzon Israel
Published: April 2020

Prior work shows that compared to European Americans, East Asians show an enhanced propensity to take the perspective of another person. In the current work, we tested whether this cultural difference might be reflected in the gray matter (GM) volume of the temporoparietal junction (TPJ), a brain region selectively implicated in perspective taking and mentalizing. We also explored whether the cultural difference in the TPJ GM volume might be moderated by dopamine D4 receptor gene (DRD4) exon 3 variable-number tandem repeat polymorphism. Structural magnetic resonance imaging of 66 European Americans and 66 East Asian-born Asians were subjected to voxel-based morphometry. It was observed that the GM volume of the right TPJ was greater among East Asians than among European Americans. Moreover, this cultural difference was significantly more pronounced among carriers of the 7- or 2-repeat allele of DRD4 than among the non-carriers of these alleles. Our findings contribute to the growing evidence that culture can shape the brain.
Effect of artificial dawn light on cardiovascular function, alertness, and balance in middle-aged and older adults
Authors: Gabel Virginie , Miglis Mitchell , Zeitzer Jamie M
Published: April 2020

Study Objectives

When arising in the morning, many older people experience dizziness and difficulty maintaining proper balance, as the cardiovascular system is not able to compensate to the postural shift (standing) and maintain sufficient blood flow to the brain. Such changes in cardiovascular function are observed in young individuals exposed to a dawn simulation light. In this study, we examined whether exposure to a dawn simulation light could impact cardiovascular function and consequent changes in balance in middle-aged and older adults.

Methods

Twenty-three participants (67.3 ± 8.8 y), 12 of whom reported a history of dizziness in the morning, underwent two overnight stays in our laboratory. During both nights, they slept in complete darkness, except for the last 30 minutes of one of the nights during which a dawn simulation light was used. Continuous blood pressure (BP) and heart rate (HR) were monitored. Subjective and objective alertness, salivary cortisol, and mobile and standing balance were examined upon arising.

Results

Dawn simulation light decreased (33%) the amount of sleep before morning awakening, lowered BP (6.24 mmHg), and increased HR (0.93 bpm). Despite these changes in physiology, there was no significant impact of dawn simulation on subjective or objective alertness, measures of standing or ambulatory balance, morning cortisol awakening response, or cardiovascular function after awakening.

Conclusion

While the dawn simulation did cause an increase in wake and a change in cardiovascular function prior to morning arousal in older adults, we could find no evidence of a functional change in either cardiovascular function or balance upon standing.

Clinical Trial

Registered on Clinicaltrials.gov, #NCT02632318, https://clinicaltrials.gov/ct2/show/NCT02632318
Improved Genome Sequence and Gene Annotation Resource for the Potato Late Blight Pathogen Phytophthora infestans
Authors: Lee Yoonyoung , Cho Kwang-Soo , Seo Jin-Hee , Sohn Kee Hoon , Prokchorchik Maxim
Published: April 2020

Phytophthora infestans is a devastating pathogen causing potato late blight (Solanum tuberosum). Here we report the sequencing, assembly and genome annotation for two Phytophthora infestans isolates sampled in Republic of Korea. Genome sequencing was carried out using long read (Oxford Nanopore) and short read (Illumina Nextseq) sequencing technologies that significantly improved the contiguity and quality of P. infestans genome assembly. Our resources would help researchers better understand the molecular mechanisms by which P. infestans causes late blight disease in the future.
A Chromosome-Scale Genome Assembly Resource for Myriosclerotinia sulcatula Infecting Sedge Grass (Carex sp.)
Authors: Kusch Stefan , Ibrahim Heba M. M. , Zanchetta Catherine , Lopez-Roques Celine , Donnadieu Cecile , Raffaele Sylvain
Published: April 2020

The fungus Myriosclerotinia sulcatula is a close relative of the notorious polyphagous plant pathogens Botrytis cinerea and Sclerotinia sclerotiorum but exhibits a host range restricted to plants from the Carex genus (Cyperaceae family). To date, there are no genomic resources available for fungi in the Myriosclerotinia genus. Here, we present a chromosome-scale reference genome assembly for M. sulcatula. The assembly contains 24 contigs with a total length of 43.53 Mbp, with scaffold N50 of 2,649.7 kbp and N90 of 1,133.1 kbp. BRAKER-predicted gene models were manually curated using WebApollo, resulting in 11,275 protein-coding genes that we functionally annotated. We provide a high-quality reference genome assembly and annotation for M. sulcatula as a resource for studying evolution and pathogenicity in fungi from the Sclerotiniaceae family.
Sources, Spectrum, Genetics, and Inheritance ofPhaseolus vulgarisResistance AgainstXanthomonas citripv.fuscans
Authors: Monteiro Ana Leticia Rocha , Chaves Fabiana Salomão , Pantaleão Augusto Soares Lins , Carneiro Pedro Crescêncio Souza , de Souza Carneiro José Eustáquio , Badel Jorge Luis
Published: April 2020

Common bean (Phaseolus vulgaris) is one of the most consumed agricultural products in the world. Its production is affected by common bacterial blight (CBB) caused by Xanthomonas citri pv. fuscans and X. phaseoli pv. phaseoli. In this work, we investigated the spectrum, genetics, and inheritance of common bean resistance to X. citri pv. fuscans. Inoculation of nine selected cultivars with an X. citri pv. fuscans strain showed that BRS Radiante and IAPAR 16 were resistant. These two cultivars were also resistant to six X. phaseoli pv. phaseoli strains of different geographic origins, demonstrating their broad-spectrum resistances. BRS Radiante sustained smaller X. citri pv. fuscans populations than two susceptible cultivars. Stomatal densities of IAPAR 16 and BRS Radiante were significantly higher than or not different from susceptible cultivars. BRS Radiante showed the lowest general combining ability values and the combination BRS Radiante × Carioca MG the lowest specific combining ability (SCA) values, revealing the capacity of BRS Radiante to increase resistance to X. citri pv. fuscans. Positive and negative parental SCA values indicated dominant and recessive genes involved in X. citri pv. fuscans resistance. Resistance of the BRS Radiante × Carioca MG cross segregated in a 9:7 ratio in the F2population, indicating that it is governed by two complementary dominant genes. Maximum likelihood analysis showed that the resistance of BRS Radiante to X. citri pv. fuscans is conferred by a gene of major effect with contribution of additional polygenes. This study contributes with important knowledge on the resistance against CBB in Brazilian common bean cultivars as well as with molecular tools for confirmation of common bean hybrids.
Osmotin-Like Protein Gene from Panax notoginseng Is Regulated by Jasmonic Acid and Involved in Defense Responses to Fusarium solani
Authors: Zhao Qin , Qiu Bingling , Li Shan , Zhang Yingpeng , Cui Xiuming , Liu Diqiu
Published: April 2020

Osmotin and osmotin-like proteins (OLPs) play important roles in plant defense responses. The full-length cDNA sequence of an OLP gene was cloned from Panax notoginseng using rapid amplification of cDNA-end technology and named PnOLP1. A quantitative reverse transcription-PCR analysis showed that the signaling molecules methyl jasmonate, salicylic acid, ethylene, and hydrogen peroxide induced PnOLP1 expression to different degrees. In addition, the expression level of PnOLP1 rapidly increased within 48 h of inoculating P. notoginseng with the root rot pathogen Fusarium solani. Subcellular localization revealed that PnOLP1 localized to the cell wall. A prokaryotic expression vector containing PnOLP1 was constructed and transformed into Escherichia coli BL21 (DE3), and in vitro antifungal assays were performed using the purified recombinant PnOLP1 protein. The recombinant PnOLP1 protein had strong inhibitory effects on the mycelial growth of F. oxysporum, F. graminearum, and F. solani. A plant PnOLP1-overexpression vector was constructed and transfected into tobacco, and the resistance of T2 transgenic tobacco against F. solani was significantly enhanced compared with wild-type tobacco. Moreover, a PnOLP1 RNAi vector was constructed and transferred to the P. notoginseng leaves for transient expression, and the decrease of PnOLP1 expression level in P. notoginseng leaves increased the susceptibility to F. solani. Thus, PnOLP1 is an important disease resistance gene involved in the defense responses of P. notoginseng to F. solani.
Elucidating CCRK’s function in ciliary assembly and cilium‐dependent signaling
Authors: Eggenschwiler Jonathan T
Published: April 2020

Our studies are focused on the questions of how ciliary length and ciliary import of cell signaling regulators are controlled. Despite many advances, these questions have yet to be fully answered. One clue as to the mechanistic basis of these processes comes from studies on Cell Cycle‐Related Kinase (CCRK). Work on the Clamydomonas homolog, LF2, indicates that this kinase ensures that flagella are neither too long nor too short, likely mediated by regulated transport of ciliary tubulin (
Craft et al., 2015). We previously showed that mammalian CCRK is similarly required for ciliary length control and that it is needed for cells to achieve appropriate responses to Hedgehog (Hh) signals (
Snouffer et al., 2017). Additional work indicated that CCRK promotes the efficient transport of Hh pathway regulatory factors, such as Smoothened and Gli2, into cilia. However, the manner by which the kinase directs these processes is unknown. One target of CCRK phosphorylation is the ciliary length regulator Intestinal Cell Kinase (ICK); CCRK‐mediated phosphorylation in ICK’s TDY motif promotes ICK activity. Our analysis of Ccrk/Ick double mutants indicates that CCRK must also function via ICK‐independent mechanisms and that ICK promotes functions in the absence of CCRK‐mediated phosphorylation. To gain insight into the ICK‐independent CCRK functions in ciliary length control and ciliary cargo import, we performed phosphoproteomic screens for potential mediators of CCRK function. This screen has pointed to candidate mediators that regulate microfilament and microtubule dynamics. Consistent with a role for CCRK’s action via microfilament dynamics, Ccrk mutant, but not wild‐type, cells showed greatly reduced import of ciliary cargo upon treatment with sub‐threshold doses of the actin polymerization inhibitor Cytochalasin D. Our ongoing work on this mechanism will be presented.

Support or Funding Information

This work was supported by National Institute of Child Health and Human Development grant number R01HD050761 and by the Office of the Vice President of Research at the University of Georgia.
Somatotrope Responses to Acute and Prolonged Loss of Leptin Signaling
Authors: Miles Tiffany K. , Allensworth-James Melody , Moreira Ana Rita Silva , Odle Angela , Haney Anessa C. , Lagasse Alexandra , MacNicol Angus , Childs Gwen V.
Published: April 2020

In mice and humans, fasting results in a rise of serum growth hormone (GH). In mice, this coincides with increased Gh and growth hormone releasing hormone receptor (Ghrhr) mRNA. Fasting also results in increased serum ghrelin and decreased serum leptin. We have shown that pituitary somatotropes are dependent on circulating leptin to maintain sufficient stores of GH and GHRHR proteins. We hypothesized that the GH rise seen in fasted mice with reduced serum leptin was due to ghrelin‐stimulated pathways. However, a review of the literature revealed a study by Steyn et al. (2011) which demonstrated an acute decline in GH pulses in male mice after 12–18 h of fasting. This study suggests that somatotropes may have time‐dependent responses, with shorter fasting responses driven by the absence of leptin signals.

Our fasting study was composed of three groups of male mice: 1) mice fasted for 6, 12, 24, or 48 hours, 2) mice fasted for 24 and 48 hours with 10% glucose in the drinking water, and 3) mice fed ad libitum. Body weight and serum glucose were significantly reduced with all fasting times. After 6 h of fasting, serum leptin levels were reduced from 9625.63 pg/ml (fed controls) to 1798.15 pg/mL with a further reduction to 774.17 pg/ml after 12 h, which was maintained following 24 and 48 h fasting. Ghrelin levels were unchanged with 6, 12 and 24 h fasting but significantly increased by 55.4% with 48 h fasting. Serum GH was unchanged with 6 and 12 h fasting, but was significantly reduced from 7765 pg/ml (fed) to 2253 pg/ml after the 24 h fast (p=0.03 ANOVA). In the 48 h fasted mice, serum GH was significantly increased over fed mice, from 1828 pg/ml (fed) to 10569 pg/ml (p=0.01 ANOVA). There were significant increases in pituitary Ghrhr mRNA after 24 and 48 h fasting and Gh mRNA was increased after 6 and 48 h of fasting. However, GH and GHRHR content were unchanged.

This study has uncovered time‐dependent differential responses to fasting by somatotropes in male mice. The reduced serum GH after 24 h correlated well with the reduction in serum leptin. Similarly, the prolonged fasting (48 h) and elevated serum GH correlated well with the rise in ghrelin. The high Gh and Ghrhr mRNA levels corresponds with rising ghrelin levels. Reduced serum GH at 24 h indicates that somatotropes remain dependent on leptin signals for GH secretion. However, with normal GH or GHRHR content, this points to a defect in a signaling pathway needed for secretion that has yet to be determined.

Support or Funding Information

NIH grants 5 R01 HD087057‐03 (GVC and AM), 5 R01 HD 093461‐01 (AM and GVC), and 5 R01 DK113776‐02 (GVC and AM). NIGMS grants P20 GM103425 and P30GM11070 (PI Dr. Edgar Garcia‐Rill). NSF funded Southern Regional Education Board Dissertation Award (TKM).
Recruitment of kindlin‐3 to plasma membrane through its PH domain precedes high affinity β2 integrin activation and neutrophil arrest
Authors: Wen Lai , Marki Alex , Roy Payel , McArdle Sara , Sun Hao , Fan Zhichao , Gingras Alexandre R. , Ginsberg Mark H. , Ley Klaus
Published: April 2020

Leukocytes including neutrophils must adhere to the vascular endothelium in order to respond to injury or infection. Integrin‐mediated neutrophil adhesion starts by arrest from rolling. Activation of integrins involves conformational changes from an inactive bent conformation to an extended conformation (E+) with high affinity for ligand binding (H+). The cytoplasmic protein kindlin‐3 is required for leukocyte adhesion; mutations of kindlin‐3 cause leukocyte adhesion deficiency type III syndrome. However, the molecular mechanism by which kindlin‐3 regulates β2 integrin activation is unknown. Here we measured the spatiotemporal dynamics of kindlin‐3 and β2 integrin conformation changes during neutrophil and HL‐60 cell rolling and arrest under flow. Using high‐resolution quantitative dynamic footprinting (qDF) microscopy and kindlin‐3‐fluorescent protein (FP) fusion proteins, we find that kindlin‐3 is recruited to the plasma membrane prior to induction of the H+ β2 integrin conformation. Deletion of kindlin‐3 or its lipid‐binding pleckstrin homology (PH) domain completely abolished H+ β2 integrin induction. Intravital imaging of bone marrow chimeric mice revealed that EGFP‐tagged kindlin‐3‐expressing neutrophils, but not control kindlin‐3‐deficient neutrophils, arrested in cremaster venules in vivo in response to CXCL1. Our data show that kindlin‐3 is indispensable for the H+ β2 integrin conformation and crucial for integrin‐mediated leukocyte adhesion during inflammation.

Support or Funding Information

This work was supported by grants from the National Institutes of Health, USA (HL078784 to K.L., R01HL145454 to Z.F.), a Postdoctoral Fellowship (19POST34450228 to L.W.) and a Career Development Award (18CDA34110426 to Z.F.) from the American Heart Association, USA.
Mitochondrial RNA‐Chromatin Interactome Regulates Endothelial‐Mesenchymal Transition
Authors: Sriram Kiran , Qi Zhijie , Zhong Sheng , Chen Zhen
Published: April 2020

Endothelial cells (ECs) play a central role in vascular function and diabetes‐induced endothelial dysfunction leads to the development of cardiovascular diseases. In response to hyperglycemic and inflammatory (high‐glucose + TNFα; HT) stimuli, we found that ECs undergo dysfunction and endothelial‐mesenchymal transition (EndoMT) characterized by upregulation of inflammatory, fibrotic, and mesenchymal genes. To determine whether interaction between regulatory RNAs and chromatin contributes to the induction of EndoMT, we profiled the global changes in RNA‐chromatin interactome in HT‐treated ECs using iMARGI (in‐situ Mapping of RNA‐Genome Interactions). iMARGI revealed extensive chromatin binding of RNAs, particularly at genomic regions containing super enhancers (SEs). SE overlapping SMAD3 (a known regulator of EndoMT) becomes an active SE‐hub, while SMAD3 expression is significantly induced at the single‐cell level with HT treatment. In parallel, iMARGI revealed significant chromatin binding of mitochondrial RNAs with the degree of MT‐RNR2‐binding increasing in a time‐dependent manner with HT treatment. The most enriched SE region for MT‐RNR2‐binding also overlaps with SMAD3. Further preliminary experiments by cell fractionation verified the chromatin‐localized MT‐RNR2‐derived transcripts and their increase in response to HT treatment. Perturbation of mitochondrial function or genetic ablation of mitochondrial transcripts suggests a role for mitochondrial RNAs in regulating EC function and HT‐induced EndoMT. Collectively, our study provides novel insights into the potential involvement of mitochondrial RNAs in retrograde signaling and linking mitochondria to functional genomics important for endothelial regulation and vascular disease.

Support or Funding Information

Ella Fitzgerald Foundation, NIH grants R00HL122368, R01HL108735, R01HL145170, and NIH 4D Nucleome U01CA200147
A Novel Method for Normalization in Western Blotting that is Superior to the Use of Housekeeping Proteins
Authors: Thompson Jordan , Diller Thomas , Steer Brian
Published: April 2020

Western blotting workflows have long relied upon the use of housekeeping proteins (HKPs) as a control for sample normalization in order to account for differences between sample loads. Unfortunately, HKPs often show signal saturation, can vary in response to the treatments being studied, or show variation in expression between different cell types leading to erroneous results. In contrast, new methods of normalization that involve Total Protein Normalization (TPN) have been gaining support. In this study we investigate a new method for TPN that utilizes the Invitrogen No‐Stain Protein Labeling Reagent, which allows for all of the proteins on a membrane to be labeled uniformly and quickly while not interfering with downstream immunodetection methods. We compare this new method against common housekeeping proteins in four different cell backgrounds (HeLa, MCF‐7, Jurkat, and A431 cells). Our data illustrate how common housekeeping proteins exhibit signal saturation and yield incorrect normalization data. The No‐Stain TPN method shows superior protein normalization compared to the use of housekeeping proteins with decreased sample‐to‐sample variation (8% versus 48%). The No‐Stain TPN method provides an elegant alternative for achieving accurate quantitative western blots.

Support or Funding Information

This work was supported by Thermo Fisher Scientific
GPR120‐mediated Connexin43 Phosphorylation is Implicated in Antiarrhythmic Effects of Icosapent Ethyl in Post‐infarcted Rat Hearts
Authors: Chen Wei-Ting , Lee Chen-Che , Lee Tsung-Ming
Published: April 2020

Aims

The w‐3 fatty acids exert as an antioxidant via the G protein‐coupled receptor 120 (GPR120). Icosapent ethyl, a highly purified ethyl ester of eicosapentaenoic acid, showed a marked reduction in sudden cardiac death. Connexin43 is sensitive to redox status. We assessed whether icosapent ethyl attenuates fatal arrhythmias after myocardial infarction, a status of high oxidative stress, through increased connnexin43 expression and whether the GPR120 signaling is involved in the protection.

Methods and results

Male Wistar rats after ligating coronary artery were randomized to either vehicle or icosapent ethyl for 4 weeks. The postinfarction period was associated with increased oxidative‐nitrosative stress, as measured by myocardial superoxide, nitrotyrosine, and dihydroethidium fluorescent staining. In concert, myocardial connnexin43 levels revealed a significant decrease in vehicle‐treated infarcted rats compared with sham. These changes of oxidative‐nitrosative stress and connnexin43 levels were blunted after icosapent ethyl administration. Provocative arrhythmias in the infarcted rats treated with icosapent ethyl were significantly improved than vehicle. Icosapent ethyl significantly increased GPR120 compared to vehicle after infarction. The effects of icosapent ethyl on superoxide and connnexin43 were similar to GPR120 agonist GW9508. Besides, the effects of icosapent ethyl on oxidative‐nitrosative stress and connnexin43 phosphorylation were abolished by administering AH‐7614, an inhibitor of GPR120. SIN‐1 abolished the Cx43 phosphorylation of IPE without affecting GPR120 levels.

Conclusions

Chronic use of icosapent ethyl after infarction is associated with up‐regulation of connnexin43 phosphorylation through a GPR120‐dependent antioxidant pathway and thus plays a role in the beneficial effect on arrhythmogenic response to programmed electrical stimulation.

Support or Funding Information

This work was supported by the grants of China Medical University (DMR‐108‐035) and Ministry of Science and Technology (MOST‐108‐2622‐B‐039‐002‐CC3), Taiwan.
Skeletal Muscle Oxidative Capacity is Linked to Cardiovascular Health
Authors: Bubb Aleena M. , Blanks Anson , Looney Jacob , Blackburn Marsha , Bieck Maire Rose , Derella Cassandra C. , Sanchez Savanna , Pace Brooke , Thomas Jeffrey , Norland Kimberly , Ye Bingwei , Wang Xialong , Harris Ryan A.
Published: April 2020

Background

Cardiovascular disease (CVD) affects 48% of the adult population and is the leading cause of death in the United States. Vascular stiffness, endothelial dysfunction, and decreased skeletal muscle health are each independently associated with increased CVD risk. This study sought to test the hypothesis that indices of vascular health are associated with skeletal muscle function in the general population.

Methods

22 (16 women and 6 men) apparently healthy adults participated in this study. Dual energy x‐ray absorptiometry was performed to obtain body composition and skeletal muscle mass. Carotid‐femoral pulse wave velocity (PWVCF), augmentation index normalized for heart rate (AI@75bpm), and flow mediated dilation normalized for shear rate (FMD/Shear) were assessed to evaluate arterial stiffness and endothelial function. Handgrip strength was assessed using a handgrip dynamometer and skeletal muscle mitochondrial oxidative capacity (Kox cap) was assessed using near‐infrared spectroscopy (NIRS) to evaluate indices of skeletal muscle health and function, respectively.

Results

Kox cap was inversely correlated with both carotid‐femoral pulse wave velocity PWVCF (r= −0.515; p= 0.014) and AI@75BPM (r= −0.453; p= 0.034). In addition, a trend in the relationship between FMD/Shear and Kox cap (r= 0.326; p= 0.139) was observed. Compared with women, men had significantly higher PWVCF (p= 0.008), handgrip strength (p< 0.001), and skeletal muscle mass (p= 0.006); however, muscle function was similar (p= 0.209) between sexes.

Conclusions

These findings demonstrate that improved muscle function can favorably impact vascular health. Additionally, skeletal muscle strength and size were significantly different between sexes; however, muscle function was similar. These data highlight the importance of assessing all three indices of muscle health. Future studies are warranted to investigate if increases in vascular endothelial function will improve skeletal muscle function and contribute to prevention of CVD.

Support or Funding Information

This project was supported in part by The Augusta University Medical Scholars Program (AB) and NIH R01DK117365 (RAH and XW).
Activation of Piezo1 Mechanosensitive Channels in Retinal Ganglion Cells Leads to Cell Death
Authors: Friemel Taylor , Grillo Stephanie , Stella Salvatore L.
Published: April 2020

Purpose

Piezo1 mechanosensitive ion channels play an important role in mechanotransduction in all vertebrates and have been identified in a variety of tissues and cell types including neurons. Previously, we have identified Piezo1 mechanosensitive ion channels in zebrafish and mammalian retinal ganglion cells (RGCs). The principle aim of this study is to test whether activation of Piezo1 channels can produce neurodegeneration of zebrafish and mammalian RGCs.

Methods

For zebrafish Ca2+ imaging was performed using a genetically‐encoded Ca2+ indicator, GCaMP6f, and was targeted predominantly to RGCs using the Tg(elavl3: GCaMP6f) zebrafish line, and confirmed by backfilling the optic nerve with Texas Red hydrazide. In order to activate Piezo1 channels in RGCs, cytosolic [Ca2+]i changes were measured in response to application of the Piezo1 selective agonist, Yoda1. Immunoblotting and immunohistochemical localization for Piezo1 were used to determine the expression profile in rodent and zebrafish retina using retinal wholemounts and vertical sections. To examine apoptosis, a short‐term organotypic eyecup culture preparation was used. Eyecups were incubated in oxygenated Ringer’s solution (for 1–8 hrs) with various concentrations of Yoda1 (0.1–100μM). Following treatment with Yoda1, eyecups were fixed, sectioned, and prepared for immunohistochemistry. TUNEL assays was used to label apoptotic cells in fixed vertical retinal sections, and TUNEL‐positive cells were quantified using ImageJ software. All imaging was acquired on a Nikon C2+ confocal microscope.

Results

Calcium imaging experiments revealed that Yoda1 evokes a dose‐dependent activation of Piezo1 ion channels in GCAMP6f expressing RGCs, which was reduced in the presence of GsMTx‐4, a mechanosensitive channel antagonist. Immunohistochemical findings show strong Piezo1 immunoreactivity in RGCs and their axons in rodent and zebrafish retina. Moreover, Yoda1 evoked a concentration‐dependent increase in TUNEL‐positive labeling of RGCs in both zebrafish and rodent retina, implicating a common Ca2+‐mediated pathway for Piezo1 channel induced RGC death.

Conclusions

This study provides the first evidence for Piezo1 channel‐mediated RGC death and suggests that there is a potential link between Piezo1 channel activation on RGCs, increased [Ca2+]i influx and apoptosis. Therefore, changes in cell stiffness and pressure acting through Piezo1 mechanosensitive channels could contribute to the neurodegenerative effects observed in optic neuropathies, such as ocular injury and glaucoma.

Support or Funding Information

HMC CURE Zebrafish Pilot Grant, Penn State University‐Hershey College of Medicine; Pennsylvania Department of Health, Tobacco CURE Funds
Yes‐associated protein (YAP) in Kupffer cells enhances the production of pro‐inflammatory cytokines and promotes the development of non‐alcoholic steatohepatitis
Authors: Song Kyoungsub , Kwon Hyunjoo , Han Chang , Zhang Jinqiang , Chen Weina , Ma Wenbo , Wu Tong
Published: April 2020

Background & Aims

Yes‐associated protein (YAP) plays an important role in hepatocarcinogenesis, although the potential role of YAP in non‐neoplastic liver diseases remains largely unknown. We report herein that YAP in Kupffer cells (KCs) enhances the production of pro‐inflammatory cytokines and promotes the development of non‐alcoholic steatohepatitis.

Methods

We generated mice with Kupffer cell (KC)‐specific deletion of YAP (YAPφKO) or TLR4 (TLR4φKO) and the animals were fed high fat diet (HFD) or treated with LPS. The liver tissues were harvested and subjected to histopathological and biochemical analyses. Kupffer cells were isolated for in vitro analyses. Chromatin immunoprecipitation, DNA pull‐down, qRT‐PCR and promoter reporter activity assays were performed to validate transcriptional regulation of YAP and inflammatory cytokine production in Kupffer cells/macrophages. Analyses of liver tissues from NASH patients were performed using immunofluorescence imaging and Gene Expression Omnibus (GEO) datasets.

Results

The expression of YAP is significantly increased in KCs of wild type mice fed HFD. The YAPφKO mice fed HFD showed lower serum ALT/AST levels and less hepatic inflammation when compared to their littermate controls. LPS treatment induced the accumulation of YAP in KCs in vitro and in mice which was prevented by KC‐specific deletion of TLR4 (TLR4φKO). LPS transcriptionally activates YAP via AP‐1 in macrophages/KCs. The LPS‐induced YAP further enhances the expression of pro‐inflammatory cytokines (including MCP‐1, TNF‐α and IL‐6) through YAP association with the TEAD‐binding motif in the promoter region of inflammatory cytokines. Forced overexpression of active YAP (YAP5SA) in KCs enhanced the production of pro‐inflammatory cytokines. Treatment of HFD‐fed mice with the YAP inhibitor, verteporfin, prevented Kupffer cell activation, reduced liver inflammation and decreased serum ALT/AST levels. Analyses of liver tissues from NASH patients reveal that YAP is increased in KCs and that the level of YAP in human liver tissues is positively correlated with the expression of pro‐inflammatory cytokines.

Conclusions

This study describes an important role of YAP in Kupffer cells for regulation of liver inflammation in NASH. Our findings suggest that inhibition of YAP may represent a novel and effective therapeutic strategy for NASH treatment.
Female Offspring of Hyperandrogenemic Female (HAF) Rat Model Exhibit Insulin Resistance and Increased Blood Pressure With Aging
Authors: Shawky Noha M. , Vinson Ruth M. , Zuchowski Yvonne P. , Reckelhoff Jane F.
Published: April 2020

Background

Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenemia, oligo/an‐ovulation and polycystic ovaries. It is also associated with metabolic dysfunction and elevated blood pressure (BP). Whether daughters born to PCOS mothers exhibit a PCOS phenotype is controversial. In addition, whether daughters of PCOS women develop metabolic dysfunction and elevated BP with aging has not been reported.

Objectives

The present study aimed to test the hypothesis that aging causes an increase in MAP and metabolic dysfunction in female offspring of HAF rats (model of PCOS).

Methods

Hyperandrogenemia was induced in Sprague Dawley (SD) females by implantation of 5α‐dihydrotestosterone pellets (DHT; 7.5 mg/90 days, subcutaneously) starting at 4 weeks of age. Rats were mated with SD males, and allowed to deliver and lactate. Female offspring (F1HAF) were weighed at birth, left untreated and studied at 4–6 (adult) or 16–18 (aging) months of age. Control dams were implanted with placebo pellets subcutaneously, allowed to mate, deliver and suckle their offspring (F1Placebo).

Results

Despite the similar litter size, body weight (BW) at birth was significantly lower in female F1HAF compared to F1Placebo (6.0 ± 0.1 vs 6.5 ± 0.1 g, n=13–15). As adults (4–6 months), BW, fat and lean masses, proteinuria and mean arterial pressure (MAP, radiotelemetry) of female F1HAF were not different compared to F1Placebo. With aging, female F1HAF rats showed increased MAP compared to F1Placebo (109 ± 1 versus 96 ± 1 mmHg, n=3–4). Fasting blood glucose was not different between groups (91 ± 3 vs 90 ± 2 mg/dl), but female F1HAF rats showed significant hyperinsulinemia (0.90 ± 0.05 vs 0.78 ± 0.03 ng/ml, p ≤ 0.05) and insulin resistance (­ HOMA‐IR index; 3.3 ± 0.2 vs 2.8 ± 0.1, p ≤ 0.05, (n = 5–6)). Despite the insulin resistance, lean mass (248.4 ± 8.5 vs 241.2 ± 3.9 g) and fat mass (41.2 ± 7.9 vs 39.7 ± 1.8 g) by Echo MRI, in addition to body weight (307.5 ± 16.2 vs 298.1 ± 6.4 g; (n = 2–4)) were also not different between groups.

Conclusion

Adult female offspring of hyperandrogenemic females do not show a PCOS phenotype (e.g. no hypertension, insulin resistance, metabolic phenotype), whereas with aging, they develop hypertension, hyperinsulinemia and insulin resistance with no changes in fasting glucose, lean/fat mass or body weight. These data suggest that women whose mothers had PCOS may be at increased risk for hypertension and metabolic dysfunction with aging, although the mechanisms for the hypertension are not clear.

Support or Funding Information

R01HL135089, P01HL051971, P20GM121334, P20GM104357
Evidence for P2Y2 Receptor Facilitation of Hyperglycemia‐induced Insulin Resistance in Human Hepatocytes
Authors: Senfeld Jared I. , Shen Jianzhong
Published: April 2020

It is known that deletion of the ectonucleotidase CD39 leads to liver insulin resistance in mice; however, it remains unknown whether this phenotype is due to a lack of extracellular adenosine generation or an excessive nucleotides accumulation and signaling. We hypothesized that the nucleotide P2Y2 receptor mediates insulin resistance in human hepatocytes. RT‐PCR showed that among the eight known P2Y receptors, only the P2Y2, P2Y6 and P2Y11 mRNAs were detected in cultured HepG2 cells, a human hepatocytes model; however, stimulation of the cells with P2Y6‐ and P2Y11‐selective agonists all failed to induce intracellular Ca2+ mobilization, whereas both ATP and UTP induced significant Ca2+ signaling in a dose‐dependent manner, which was abolished by pretreatment of the cells with ARC‐118925XX, a selective and competitive P2Y2 receptor antagonist, suggesting that the P2Y2 receptor is the only functional P2Y receptor expressed in HepG2 cells. In addition, we found that stimulation of the P2Y2 receptor induced MAPK signaling including phosphorylated ERK1/2, JNK and p38, but not the AKT pathway. Interestingly, stimulation of the cells with ATP or UTP dose‐dependently blocked insulin‐induced AKT phosphorylation, but potentiated ERK1/2 signaling, indicating a selective disruption of the insulin‐AKT signaling axis by P2Y2 receptor. Furthermore, we found that a notable release of ATP into the extracellular space was observed when exposing HepG2 cells to high glucose concentrations (25mM) as compared with normal glucose (6.1 mM). Consistent with this, we also found that extracellular nucleotides signaling through P2Y2 receptor induced a dose‐dependent reduction in hepatocyte glucose uptake. We conclude that the P2Y2 receptor mediates liver insulin resistance in response to hyperglycemia, possibly through activation of the JNK pathway and inhibition of insulin signaling to the AKT pathway, highlighting that P2Y2 receptor may be a new drug target for type‐2 diabetes.

Support or Funding Information

Supported in part by NIH funding 1R01HL125279‐01A1 (JS)
Time Restricted Feeding Reduces Vascular Damage and Oxidative Stress in Male Mice on a Chronic High Fat Diet
Authors: Pati Paramita , Zhang Dingguo , Colson Jackson , Allan John Miller , Sedaka Randee , Gamble Karen L. , Bailey Shannon M. , Pollock David M. , Pollock Jennifer S.
Published: April 2020

Irregular timing of food intake is known to influence risk for cardiometabolic disease. Studies in humans and animals demonstrate that a chronic high fat diet (HFD) leads to endothelial dysfunction and cardiovascular disease. We hypothesized that restricting food availability to the active period with a HFD leads to reduced vascular damage, increased nitric oxide synthase (NOS3) activation, and reduced oxidative stress. Mice (male 8‐week old, C57BL/6J) were given a normal diet (ND; 10% fat) or HFD (45% fat) for 20 weeks ad libitum. Time restricted feeding (TRF; food availability only during the active phase between ZT12 to ZT0 or 7 PM to 7 AM) was enforced in half of the HFD mice during the final 2 weeks of feeding. At the end of the study, aortae and plasma were collected at ZT5 and ZT17 in the middle of the inactive and active periods, respectively. 24 hour food intake was similar between ad lib and TRF mice. Aortic wall thickness was significantly increased in mice on HFD compared to ND (HFD: 76±5.1 μm; ND: 59±4.6 μm; n=6, p=0.03). Interestingly, aortic fibrosis was reduced by TRF in HFD mice (HFD: 18±2.3%; HFD+TRF: 11±1.9%; n=6, p=0.04). To assess NOS3 activation, Western blotting was used to measure pNOS3(S1177) and NOS3 abundance in aortae. At ZT5 pNOS3(S1177) abundance was not affected by TRF in HFD mice (HFD: 0.5±0.3 rdu, HFD+TRF: 0.7±0.1 rdu, n=4). TRF significantly increased pNOS3(S1177) abundance in HFD mice at ZT17 (HFD: 0.5±0.2 rdu, HFD+TRF: 2.1±0.6 rdu, n=4, p=0.06). Total NOS3 expression was unaffected by TRF at both time points. During the inactive period, endothelial‐dependent relaxation to acetylcholine was significantly impaired in HFD mice and improved by TRF (ND: 93±1.4%; HFD: 57±14%; HFD+TRF: 79±4.0%, n=5–11, ND vs HFD: p=0.01). Endothelial function was not different between groups in the active period. Endothelial‐independent relaxation was similar in all groups in both inactive and active periods. Plasma nitrite, a metabolite of NO, at ZT5 was unchanged between all groups. At ZT17 plasma nitrite was significantly decreased in HFD mice compared to ND mice and unaffected by TRF (HFD: 0.6±0.06 μM; HFD+TRF: 0.7±0.03 μM; ND: 1.1±0.2 μM; n=10–11, p=0.03). 8‐isoprostane, an indirect measure of oxidative stress, was significantly reduced by TRF in HFD mice at ZT5 (HFD: 159±31 ng/mL; HFD+TRF: 60±12 ng/mL, n=4–6, p=0.01), but not different between groups at ZT17. In conclusion, these results demonstrate that restricting timing of food intake improves vascular damage and decreases oxidative stress following diet‐induced obesity in a time‐dependent manner.

Support or Funding Information

Supported by T32 DK007545, an AHA Postdoctoral Fellowship (18POST34070051) to PP, an AHA Predoctoral Fellowship (18PRE33990345) to DZ, P01 HL069999 to JSP and DMP, and P01 HL136267 to JSP, EWI, and DMP.
SAPH‐ire TFx: A Machine Learning Recommendation Model and Webtool for the Prediction of Functional Post‐Translational Modifications
Authors: English Nolan J. , Torres Matthew
Published: April 2020

Post‐translational Modifications (PTMs), chemical or proteinaceous covalent alterations to the side chains of amino acid residues in proteins, are a rapidly expanding feature class of significant importance in cell biology. Due to a high burden of experimental proof and the lack of effective means for experimentalists to prioritize PTMs by functional significance, currently less than ~2% of all PTMs have an assigned biological function. Here, we describe a new artificial neural network model, SAPH‐ire TFx for the functional prediction of experimentally observed eukaryotic PTMs. Unlike previous functional PTM prioritization models, SAPH‐ire TFx is trained to emphasize metrics that maximally capture the range of diverse feature sets comprising the functional modified eukaryotic proteome. The model of was generated through systematic evaluation of input features, model architectures, training procedures, and interpretation metrics using a 2018 training dataset of 430,750 PTMs containing 7,480 PTMs with literature‐supported evidence of biological function. The resulting model was used to classify an expanded 2019 dataset of 512,015 PTMs (12,867 known functional) containing 102,475 PTMs unencountered in the original dataset. Model output from the 2019 extended dataset was benchmarked against pre‐existing prediction models, revealing superior performance in classification of functional and/or disease‐linked PTM sites, including drawing attention to PTMs that were previously thought inconsequential. Finally, a dynamic web interface provides customizable graphical and tabular visualization of PTM and SAPH‐ire TFx data within the context of all modifications within a protein family, exposing several metrics by which important functional PTMs can be identified for investigation.