Publications by authors named "Robert J Isfort"

38 Publications

Combinations of peptides synergistically activate the regenerative capacity of skin cells in vitro.

Int J Cosmet Sci 2021 Jul 17. Epub 2021 Jul 17.

The Procter & Gamble Company, Cincinnati, Ohio, USA.

Objective: To explore synergistic effects related to skin regeneration, peptides with distinct biological mechanisms of action were evaluated in combination with different skin cell lines in the presence or absence of niacinamide (Nam). Furthermore, the synergistic responses of peptide combinations on global gene expression were compared with the changes that occur with fractional laser resurfacing treatment, a gold standard approach for skin rejuvenation, to further define optimal peptide combinations.

Methods: Microarray profiling was used to characterize the biological responses of peptide combinations (+/- Nam) relative to the individual components in epidermal keratinocyte and dermal fibroblast cell lines. Cellular functional assays were utilized to confirm the synergistic effects of peptide combinations. Bioinformatics approaches were used to link the synergistic effects of peptide combinations on gene expression to the transcriptomics of the skin rejuvenation response from fractional laser treatment.

Results: Microarray analysis of skin cells treated with peptide combinations revealed synergistic changes in gene expression compared with individual peptide controls. Bioinformatic analysis of synergy genes in keratinocytes revealed the activation of NRF2-mediated oxidative stress responses by a combination of Ac-PPYL, Pal-KTTKS and Nam. Additional analysis revealed direct downstream transcriptional targets of NRF2/ARE exhibiting synergistic regulation by this combination of materials, which was corroborated by a cellular reporter assay. NRF2-mediated oxidative stress response pathways were also found to be activated in the transcriptomics of the early skin rejuvenation response to fractional laser treatment, suggesting the importance of this biology in the early stages of tissue repair. Additionally, the second combination of peptides (pal-KT and Ac-PPYL) was found to synergistically restore cellular ATP levels that had been depleted due to the presence of ROS, indicating an additional mechanism, whereby peptide synergies may accelerate skin repair.

Conclusion: Through combinatorial synergy studies, we have identified additional in vitro skin repair mechanisms beyond the previously described functions of individual peptides and correlated these to the transcriptomics of the skin rejuvenation response of fractional laser treatment. These findings suggest that specific peptides can act together, via complementary and synergistic mechanisms, to holistically enhance the regenerative capacity of in vitro skin cells.
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http://dx.doi.org/10.1111/ics.12725DOI Listing
July 2021

Loss of C2orf69 defines a fatal autoinflammatory syndrome in humans and zebrafish that evokes a glycogen-storage-associated mitochondriopathy.

Am J Hum Genet 2021 07 25;108(7):1301-1317. Epub 2021 May 25.

Institute of Molecular and Cell Biology, A(∗)STAR, Biopolis, Singapore 138673, Singapore.

Human C2orf69 is an evolutionarily conserved gene whose function is unknown. Here, we report eight unrelated families from which 20 children presented with a fatal syndrome consisting of severe autoinflammation and progredient leukoencephalopathy with recurrent seizures; 12 of these subjects, whose DNA was available, segregated homozygous loss-of-function C2orf69 variants. C2ORF69 bears homology to esterase enzymes, and orthologs can be found in most eukaryotic genomes, including that of unicellular phytoplankton. We found that endogenous C2ORF69 (1) is loosely bound to mitochondria, (2) affects mitochondrial membrane potential and oxidative respiration in cultured neurons, and (3) controls the levels of the glycogen branching enzyme 1 (GBE1) consistent with a glycogen-storage-associated mitochondriopathy. We show that CRISPR-Cas9-mediated inactivation of zebrafish C2orf69 results in lethality by 8 months of age due to spontaneous epileptic seizures, which is preceded by persistent brain inflammation. Collectively, our results delineate an autoinflammatory Mendelian disorder of C2orf69 deficiency that disrupts the development/homeostasis of the immune and central nervous systems.
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http://dx.doi.org/10.1016/j.ajhg.2021.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8322802PMC
July 2021

Red-COLA1: a human fibroblast reporter cell line for type I collagen transcription.

Sci Rep 2020 11 12;10(1):19723. Epub 2020 Nov 12.

Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore, 138673, Singapore.

Type I collagen is a key protein of most connective tissue and its up-regulation is required for wound healing but is also involved in fibrosis. Control of expression of this collagen remains poorly understood apart from Transforming Growth Factor beta (TGF-β1)-mediated induction. To generate a sensitive, practical, robust, image-based high-throughput-compatible reporter system, we genetically inserted a short-lived fluorescence reporter downstream of the endogenous type I collagen (COL1A1) promoter in skin fibroblasts. Using a variety of controls, we demonstrate that the cell line faithfully reports changes in type I collagen expression with at least threefold enhanced sensitivity compared to endogenous collagen monitoring. We use this assay to test the potency of anti-fibrotic compounds and screen siRNAs for regulators of TGF-β1-induced type I collagen expression. We propose our reporter cell line, Red-COLA1, as a new efficient tool to study type I collagen transcriptional regulation.
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http://dx.doi.org/10.1038/s41598-020-75683-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665053PMC
November 2020

Histological and Gene Expression Analysis of the Effects of Menopause Status and Hormone Therapy on the Vaginal Introitus and Labia Majora.

J Clin Med Res 2019 Nov 29;11(11):745-759. Epub 2019 Oct 29.

The Procter & Gamble Company, Cincinnati, OH, USA.

Background: The study aimed to determine the effect of menopausal status and hormone therapy on the introitus and labia majora at the levels of histology and gene expression.

Methods: Three cohorts of 10 women each (pre-menopause, post-menopause and post-menopause + hormone therapy) were selected based on the presentation of clinical atrophy and vaginal pH. Biopsies were obtained from the introitus (fourchette) and labia majora and processed for histology and gene expression analyses with microarrays. Other data collected included self-assessed symptoms, serum estradiol, testosterone, serum hormone binding globulin and the pH of the vagina and labia majora.

Results: The introitus appears exquisitely sensitive to hormone status. Dramatic changes were observed in histology including a thinning of the epithelium in post-menopausal subjects with vaginal atrophy. Furthermore, there was differential expression of many genes that may contribute to tissue remodeling in the atrophic introitus. Levels of expression of genes associated with wound healing, angiogenesis, cell migration/locomotion, dermal structure, apoptosis, inflammation, epithelial cell differentiation, fatty acid, carbohydrate and steroid metabolism were significantly different in the cohort exhibiting atrophy of the introitus. While changes were also observed at the labia, that site was considerably less sensitive to hormone status. The gene expression changes observed at the introitus in this study were very similar to those reported previously in the atrophic vagina providing further evidence that these changes are associated with atrophy.

Conclusions: The histological and gene expression changes occurring within the introitus after menopause may contribute to the constellation of symptoms that constitute the genitourinary syndrome of menopause.
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http://dx.doi.org/10.14740/jocmr4006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879024PMC
November 2019

PyCoTools: a Python toolbox for COPASI.

Bioinformatics 2018 11;34(21):3702-3710

Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle, UK.

Motivation: COPASI is an open source software package for constructing, simulating and analyzing dynamic models of biochemical networks. COPASI is primarily intended to be used with a graphical user interface but often it is desirable to be able to access COPASI features programmatically, with a high level interface.

Results: PyCoTools is a Python package aimed at providing a high level interface to COPASI tasks with an emphasis on model calibration. PyCoTools enables the construction of COPASI models and the execution of a subset of COPASI tasks including time courses, parameter scans and parameter estimations. Additional 'composite' tasks which use COPASI tasks as building blocks are available for increasing parameter estimation throughput, performing identifiability analysis and performing model selection. PyCoTools supports exploratory data analysis on parameter estimation data to assist with troubleshooting model calibrations. We demonstrate PyCoTools by posing a model selection problem designed to show case PyCoTools within a realistic scenario. The aim of the model selection problem is to test the feasibility of three alternative hypotheses in explaining experimental data derived from neonatal dermal fibroblasts in response to TGF-β over time. PyCoTools is used to critically analyze the parameter estimations and propose strategies for model improvement.

Availability And Implementation: PyCoTools can be downloaded from the Python Package Index (PyPI) using the command 'pip install pycotools' or directly from GitHub (https://github.com/CiaranWelsh/pycotools). Documentation at http://pycotools.readthedocs.io.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6198863PMC
November 2018

Age-induced and photoinduced changes in gene expression profiles in facial skin of Caucasian females across 6 decades of age.

J Am Acad Dermatol 2018 Jan 14;78(1):29-39.e7. Epub 2017 Nov 14.

The Procter & Gamble Company, Cincinnati, Ohio.

Background: Intrinsic and extrinsic factors, including ultraviolet irradiation, lead to visible signs of skin aging.

Objective: We evaluated molecular changes occurring in photoexposed and photoprotected skin of white women 20 to 74 years of age, some of whom appeared substantially younger than their chronologic age.

Methods: Histologic and transcriptomics profiling were conducted on skin biopsy samples of photoexposed (face and dorsal forearm) or photoprotected (buttocks) body sites from 158 women. 23andMe genotyping determined genetic ancestry.

Results: Gene expression and ontologic analysis revealed progressive changes from the 20s to the 70s in pathways related to oxidative stress, energy metabolism, senescence, and epidermal barrier; these changes were accelerated in the 60s and 70s. The gene expression patterns from the subset of women who were younger-appearing were similar to those in women who were actually younger.

Limitations: Broader application of these findings (eg, across races and Fitzpatrick skin types) will require further studies.

Conclusions: This study demonstrates a wide range of molecular processes in skin affected by aging, providing relevant targets for improving the condition of aging skin at different life stages and defining a molecular pattern of epidermal gene expression in women who appear younger than their chronologic age.
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http://dx.doi.org/10.1016/j.jaad.2017.09.012DOI Listing
January 2018

A systems approach to understanding human rhinovirus and influenza virus infection.

Virology 2015 Dec 6;486:146-57. Epub 2015 Oct 6.

The Institute for Systems Biology, Seattle, WA 98109, USA. Electronic address:

Human rhinovirus and influenza virus infections of the upper airway lead to colds and the flu and can trigger exacerbations of lower airway diseases including asthma and chronic obstructive pulmonary disease. Novel diagnostic and therapeutic targets are still needed to differentiate between the cold and the flu, since the clinical course of influenza can be severe while that of rhinovirus is usually more mild. In our investigation of influenza and rhinovirus infection of human respiratory epithelial cells, we used a systems approach to identify the temporally changing patterns of host gene expression from these viruses. After infection of human bronchial epithelial cells (BEAS-2B) with rhinovirus, influenza virus or co-infection with both viruses, we studied the time-course of host gene expression changes over three days. We modeled host responses to these viral infections with time and documented the qualitative and quantitative differences in innate immune activation and regulation.
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http://dx.doi.org/10.1016/j.virol.2015.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111289PMC
December 2015

Quantitative proteogenomic profiling of epidermal barrier formation in vitro.

J Dermatol Sci 2015 Jun 14;78(3):173-80. Epub 2015 Mar 14.

Institute for Systems Biology, 401 Terry Ave N., Seattle, WA 98109, USA. Electronic address:

Background: The barrier function of the epidermis is integral to personal well-being, and defects in the skin barrier are associated with several widespread diseases. Currently there is a limited understanding of system-level proteomic changes during epidermal stratification and barrier establishment.

Objective: Here we report the quantitative proteogenomic profile of an in vitro reconstituted epidermis at three time points of development in order to characterize protein changes during stratification.

Methods: The proteome was measured using data-dependent "shotgun" mass spectrometry and quantified with statistically validated label-free proteomic methods for 20 replicates at each of three time points during the course of epidermal development.

Results: Over 3600 proteins were identified in the reconstituted epidermis, with more than 1200 of these changing in abundance over the time course. We also collected and discuss matched transcriptomic data for the three time points, allowing alignment of this new dataset with previously published characterization of the reconstituted epidermis system.

Conclusion: These results represent the most comprehensive epidermal-specific proteome to date, and therefore reveal several aspects of barrier formation and skin composition. The limited correlation between transcript and protein abundance underscores the importance of proteomic analysis in developing a full understanding of epidermal maturation.
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http://dx.doi.org/10.1016/j.jdermsci.2015.02.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424144PMC
June 2015

Mouse hair cycle expression dynamics modeled as coupled mesenchymal and epithelial oscillators.

PLoS Comput Biol 2014 Nov 6;10(11):e1003914. Epub 2014 Nov 6.

Institute for Systems Biology, Seattle, Washington, United States of America.

The hair cycle is a dynamic process where follicles repeatedly move through phases of growth, retraction, and relative quiescence. This process is an example of temporal and spatial biological complexity. Understanding of the hair cycle and its regulation would shed light on many other complex systems relevant to biological and medical research. Currently, a systematic characterization of gene expression and summarization within the context of a mathematical model is not yet available. Given the cyclic nature of the hair cycle, we felt it was important to consider a subset of genes with periodic expression. To this end, we combined several mathematical approaches with high-throughput, whole mouse skin, mRNA expression data to characterize aspects of the dynamics and the possible cell populations corresponding to potentially periodic patterns. In particular two gene clusters, demonstrating properties of out-of-phase synchronized expression, were identified. A mean field, phase coupled oscillator model was shown to quantitatively recapitulate the synchronization observed in the data. Furthermore, we found only one configuration of positive-negative coupling to be dynamically stable, which provided insight on general features of the regulation. Subsequent bifurcation analysis was able to identify and describe alternate states based on perturbation of system parameters. A 2-population mixture model and cell type enrichment was used to associate the two gene clusters to features of background mesenchymal populations and rapidly expanding follicular epithelial cells. Distinct timing and localization of expression was also shown by RNA and protein imaging for representative genes. Taken together, the evidence suggests that synchronization between expanding epithelial and background mesenchymal cells may be maintained, in part, by inhibitory regulation, and potential mediators of this regulation were identified. Furthermore, the model suggests that impairing this negative regulation will drive a bifurcation which may represent transition into a pathological state such as hair miniaturization.
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http://dx.doi.org/10.1371/journal.pcbi.1003914DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4222602PMC
November 2014

Unique Responses are Observed in Transient Receptor Potential Ankyrin 1 and Vanilloid 1 (TRPA1 and TRPV1) Co-Expressing Cells.

Cells 2014 Jun 11;3(2):616-26. Epub 2014 Jun 11.

Cardiovascular and Respiratory Studies, The University of Hull, HU6 7RX, UK.

Transient receptor potential (TRP) ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors are implicated in modulation of cough and nociception. In vivo, TRPA1 and TRPV1 are often co-expressed in neurons and TRPA1V1 hetero-tetramer formation is noted in cells co-transfected with the respective expression plasmids. In order to understand the impact of TRP receptor interaction on activity, we created stable cell lines expressing the TRPA1, TRPV1 and co-expressing the TRPA1 and TRPV1 (TRPA1V1) receptors. Among the 600 compounds screened against these receptors, we observed a number of compounds that activated the TRPA1, TRPV1 and TRPA1V1 receptors; compounds that activated TRPA1 and TRPA1V1; compounds that activated TRPV1 and TRPA1V1; compounds in which TRPA1V1 response was modulated by either TRPA1 or TRPV1; and compounds that activated only TRPV1 or TRPA1 or TRPA1V1; and one compound that activated TRPA1 and TRPV1, but not TRPA1V1. These results suggest that co-expression of TRPA1 and TRPV1 receptors imparts unique activation profiles different from that of cells expressing only TRPA1 or TRPV1.
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http://dx.doi.org/10.3390/cells3020616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4092848PMC
June 2014

Transcriptional profiling of epidermal barrier formation in vitro.

J Dermatol Sci 2014 Mar 11;73(3):187-97. Epub 2013 Nov 11.

The Procter & Gamble Company, Mason Business Center, Cincinnati, OH 45040, USA. Electronic address:

Background: Barrier function is integral to the health of epithelial tissues. Currently, there is a broad need to develop and improve our knowledge with regard to barrier function for reversal of mild skin irritation and dryness. However, there are few in vitro models that incorporate modulations of both lipids and epidermal differentiation programs for pre-clinical testing to aid in the understanding of barrier health.

Objective: We have generated a reconstituted epidermis on a decellularized dermis (DED) and characterized its barrier properties relative to human epidermis in order to determine its utility for modeling barrier formation and repair.

Methods: We followed the process of epidermal differentiation and barrier formation through immunocytochemistry and transcriptional profiling. We examined barrier functionality through measurements of surface pH, lipid composition, stratum corneum water content, and the ability to demonstrate topical dose-dependent exclusion of surfactant.

Results: Transcriptional profiling of the epidermal model during its formation reveals temporal patterns of gene expression associated with processes regulating barrier function. The profiling is supported by gradual formation and maturation of a stratum corneum and expression of appropriate markers of epidermis development. The model displays a functional barrier and a water gradient between the stratum corneum and viable layers, as determined by confocal Raman spectroscopy. The stratum corneum layer displays a normal acidic pH and an appropriate composition of barrier lipids.

Conclusion: The epidermal model demonstrates its utility as an investigative tool for barrier health and provides a window into the transcriptional regulation of multiple aspects of barrier formation.
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http://dx.doi.org/10.1016/j.jdermsci.2013.11.004DOI Listing
March 2014

A differential pattern of gene expression in skeletal muscle of tumor-bearing rats reveals dysregulation of excitation–contraction coupling together with additional muscle alterations.

Muscle Nerve 2014 Feb;49(2):233-48

Introduction: Cachexia is a wasting condition that manifests in several types of cancer. The main characteristic of this condition is a profound loss of muscle mass.

Methods: By using a microarray system, expression of several hundred genes was screened in skeletal muscle of rats bearing a cachexia-inducing tumor, the AH-130 Yoshida ascites hepatoma. This model induced a strong decrease in muscle mass in the tumor-bearing animals, as compared with their healthy counterparts.

Results: The results show important differences in gene expression in EDL skeletal muscle between tumor-bearing animals with cachexia and control animals.

Conclusions: The differences observed pertain to genes related to intracellular calcium homeostasis and genes involved in the control of mitochondrial oxidative phosphorylation and protein turnover, both at the level of protein synthesis and proteolysis. Assessment of these differences may be a useful tool for the design of novel therapeutic strategies to fight this devastating syndrome.
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http://dx.doi.org/10.1002/mus.23893DOI Listing
February 2014

Activation of the dopamine 1 and dopamine 5 receptors increase skeletal muscle mass and force production under non-atrophying and atrophying conditions.

BMC Musculoskelet Disord 2011 Jan 26;12:27. Epub 2011 Jan 26.

Research Division, Procter & Gamble, Mason, OH, USA.

Background: Control of skeletal muscle mass and force production is a complex physiological process involving numerous regulatory systems. Agents that increase skeletal muscle cAMP levels have been shown to modulate skeletal muscle mass and force production. The dopamine 1 receptor and its closely related homolog, the dopamine 5 receptor, are G-protein coupled receptors that are expressed in skeletal muscle and increase cAMP levels when activated. Thus we hypothesize that activation of the dopamine 1 and/or 5 receptor will increase skeletal muscle cAMP levels thereby modulating skeletal muscle mass and force production.

Methods: We treated isolated mouse tibialis anterior (TA) and medial gastrocnemius (MG) muscles in tissue bath with the selective dopamine 1 receptor and dopamine 5 receptor agonist SKF 81297 to determine if activation of skeletal muscle dopamine 1 and dopamine 5 receptors will increase cAMP. We dosed wild-type mice, dopamine 1 receptor knockout mice and dopamine 5 receptor knockout mice undergoing casting-induced disuse atrophy with SKF 81297 to determine if activation of the dopamine 1 and dopamine 5 receptors results in hypertrophy of non-atrophying skeletal muscle and preservation of atrophying skeletal muscle mass and force production.

Results: In tissue bath, isolated mouse TA and MG muscles responded to SKF 81297 treatment with increased cAMP levels. Treating wild-type mice with SKF 81297 reduced casting-induced TA and MG muscle mass loss in addition to increasing the mass of non-atrophying TA and MG muscles. In dopamine 1 receptor knockout mice, extensor digitorum longus (EDL) and soleus muscle mass and force was not preserved during casting with SKF 81297 treatment, in contrast to significant preservation of casted wild-type mouse EDL and soleus mass and EDL force with SKF 81297 treatment. Dosing dopamine 5 receptor knockout mice with SKF 81297 did not significantly preserve EDL and soleus muscle mass and force although wild-type mouse EDL mass and force was significantly preserved SKF 81297 treatment.

Conclusions: These data demonstrate for the first time that treatment with a dopamine 1/5 receptor agonist results in (1) significant preservation of EDL, TA, MG and soleus muscle mass and EDL muscle force production during periods of atrophy and (2) hypertrophy of TA and MG muscle. These effects appear to be mainly mediated by both the dopamine 1 and dopamine 5 receptors.
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http://dx.doi.org/10.1186/1471-2474-12-27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3038169PMC
January 2011

Treatment with a corticotrophin releasing factor 2 receptor agonist modulates skeletal muscle mass and force production in aged and chronically ill animals.

BMC Musculoskelet Disord 2011 Jan 14;12:15. Epub 2011 Jan 14.

Research Division, Procter & Gamble Company, Mason, OH, USA.

Background: Muscle weakness is associated with a variety of chronic disorders such as emphysema (EMP) and congestive heart failure (CHF) as well as aging. Therapies to treat muscle weakness associated with chronic disease or aging are lacking. Corticotrophin releasing factor 2 receptor (CRF2R) agonists have been shown to maintain skeletal muscle mass and force production in a variety of acute conditions that lead to skeletal muscle wasting.

Hypothesis: We hypothesize that treating animals with a CRF2R agonist will maintain skeletal muscle mass and force production in animals with chronic disease and in aged animals.

Methods: We utilized animal models of aging, CHF and EMP to evaluate the potential of CRF2R agonist treatment to maintain skeletal muscle mass and force production in aged animals and animals with CHF and EMP.

Results: In aged rats, we demonstrate that treatment with a CRF2R agonist for up to 3 months results in greater extensor digitorum longus (EDL) force production, EDL mass, soleus mass and soleus force production compared to age matched untreated animals. In the hamster EMP model, we demonstrate that treatment with a CRF2R agonist for up to 5 months results in greater EDL force production in EMP hamsters when compared to vehicle treated EMP hamsters and greater EDL mass and force in normal hamsters when compared to vehicle treated normal hamsters. In the rat CHF model, we demonstrate that treatment with a CRF2R agonist for up to 3 months results in greater EDL and soleus muscle mass and force production in CHF rats and normal rats when compared to the corresponding vehicle treated animals.

Conclusions: These data demonstrate that the underlying physiological conditions associated with chronic diseases such as CHF and emphysema in addition to aging do not reduce the potential of CRF2R agonists to maintain skeletal muscle mass and force production.
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http://dx.doi.org/10.1186/1471-2474-12-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3025927PMC
January 2011

Patterns of gene expression in muscle and fat in tumor-bearing rats: effects of CRF2R agonist on cachexia.

Muscle Nerve 2010 Dec;42(6):936-49

Cancer Research Group, Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, Barcelona 08028, Spain.

The hypothesis we tested was that administering corticotropin-releasing factor receptor agonists preserves muscle mass during cancer that is related to changes in tissue gene expression. cDNA microarrays were used to compare mRNAs from muscle and adipose tissues of non-treated and agonist-treated tumor-bearing rats. In muscle of non-tumor-bearing agonist-treated animals we observed decreased expression of genes associated with fatty acid uptake and esterification. In tumor-bearing animals, CRF2R agonist administration produced decreased mRNA content of the atrogene lipin-1. In white adipose tissue, agonist treatment of non-tumor-bearing animals induced genes typically related to muscle structure and function. The fact that this treatment decreased expression of atrogenes could have clinical application. In addition, agonist treatment changed the gene pattern of adipose tissue to render it similar to that of skeletal muscle; thus, treatment with this agonist alters the gene pattern to what could be called "muscularization of white adipose tissue."
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http://dx.doi.org/10.1002/mus.21781DOI Listing
December 2010

Limb immobilization induces a coordinate down-regulation of mitochondrial and other metabolic pathways in men and women.

PLoS One 2009 Aug 5;4(8):e6518. Epub 2009 Aug 5.

Department of Pediatrics & Medicine, McMaster University, Hamilton, Ontario, Canada.

Advancements in animal models and cell culture techniques have been invaluable in the elucidation of the molecular mechanisms that regulate muscle atrophy. However, few studies have examined muscle atrophy in humans using modern experimental techniques. The purpose of this study was to examine changes in global gene transcription during immobilization-induced muscle atrophy in humans and then explore the effects of the most prominent transcriptional alterations on protein expression and function. Healthy men and women (N = 24) were subjected to two weeks of unilateral limb immobilization, with muscle biopsies obtained before, after 48 hours (48 H) and 14 days (14 D) of immobilization. Muscle cross sectional area (approximately 5%) and strength (10-20%) were significantly reduced in men and women (approximately 5% and 10-20%, respectively) after 14 D of immobilization. Micro-array analyses of total RNA extracted from biopsy samples at 48 H and 14 D uncovered 575 and 3,128 probes, respectively, which were significantly altered during immobilization. As a group, genes involved in mitochondrial bioenergetics and carbohydrate metabolism were predominant features at both 48 H and 14 D, with genes involved in protein synthesis and degradation significantly down-regulated and up-regulated, respectively, at 14 D of muscle atrophy. There was also a significant decrease in the protein content of mitochondrial cytochrome c oxidase, and the enzyme activity of cytochrome c oxidase and citrate synthase after 14 D of immobilization. Furthermore, protein ubiquitination was significantly increased at 48 H but not 14 D of immobilization. These results suggest that transcriptional and post-transcriptional suppression of mitochondrial processes is sustained throughout 14 D of immobilization, while protein ubiquitination plays an early but transient role in muscle atrophy following short-term immobilization in humans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006518PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2716517PMC
August 2009

Sex differences in global mRNA content of human skeletal muscle.

PLoS One 2009 Jul 22;4(7):e6335. Epub 2009 Jul 22.

Department of Medical Sciences, McMaster University, Hamilton, Ontario, Canada.

Women oxidize more fat as compared to men during endurance exercise and several groups have shown that the mRNA content of selected genes related to fat oxidation are higher in women (e.g. hormone sensitive lipase, beta-hydroxyacyl-CoA dehydrogenase, CD36). One of the possible mechanisms is that women tend to have a higher area percentage of type I skeletal muscle fibers as compared with men. Consequently, we hypothesized that sex would influence the basal mRNA and protein content for genes involved in metabolism and the determination of muscle fiber type. Muscle biopsies from the vastus lateralis were collected from healthy men and women. We examined mRNA content globally using Affymetrix GeneChips, and selected genes were examined and/or confirmed by RT-PCR. Furthermore, we examined protein content by Western blot analysis. Stringent gene array analysis revealed 66 differentially expressed genes representing metabolism, mitochondrial function, transport, protein biosynthesis, cell proliferation, signal transduction pathways, transcription and translation. Stringent gene array analysis and RT-PCR confirmed that mRNA for; acyl-coenzyme A acyltransferase 2 (ACAA2), trifunctional protein beta (HADHB), catalase, lipoprotein lipase (LPL), and uncoupling protein-2 (UCP-2) were higher in women. Targeted gene analysis revealed that myosin heavy chain I (MHCI), peroxisome proliferator-activated receptor (PPAR)delta were higher in women compared with men. Surprisingly, there were no significant sex based differences in protein content for HADHB, ACAA2, catalase, PPARdelta, and MHC1. In conclusion, the differences in the basal mRNA content in resting skeletal muscle suggest that men and women are transcriptionally "primed" for known physiological differences in metabolism however the mechanism behind sex differences in fiber type remains to be determined.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0006335PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2709437PMC
July 2009

Effects of CRF2R agonist on tumor growth and cachexia in mice implanted with Lewis lung carcinoma cells.

Muscle Nerve 2008 Feb;37(2):190-5

Cancer Research Group, Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, Barcelona 08028, Spain.

Previous studies have demonstrated an effect of corticotropin-releasing factor 2 receptor (CRF2R) agonists in the maintenance of skeletal muscle mass. The aim of this study was to evaluate the effects of a CRF2R agonist in preserving skeletal muscle in a mouse cachexia model. Implantation of a fast-growing tumor to mice (Lewis lung carcinoma) resulted in a clear cachectic state characterized by a profound muscle wasting. We found that administration of a CRF2R agonist (PG-873637) at 100 microg/kg/day by means of osmotic minipumps to tumor-bearing mice resulted in beneficial effects on muscle weight loss. Thus, muscle loss was partially reversed by the CRF2R agonist at different stages of tumor growth (at day 14 after tumor inoculation: 12% in tibialis posterior; 9% in gastrocnemius; and 48% in soleus). Moreover, the CRF2R agonist significantly reduced both the number of metastases and their mass (at day 19 after tumor inoculation: 66% and 61%, respectively). These data suggest a potentially beneficial effect of the CRF2R agonist in preserving skeletal muscle during cancer cachexia and open a line of research for the development of new therapeutic approaches for the treatment of muscle wasting associated with cancer.
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http://dx.doi.org/10.1002/mus.20899DOI Listing
February 2008

Corticortophin releasing factor 2 receptor agonist treatment significantly slows disease progression in mdx mice.

BMC Med 2007 Jul 12;5:18. Epub 2007 Jul 12.

Research Division, Procter & Gamble Pharmaceuticals, Mason, OH, USA.

Background: Duchenne muscular dystrophy results from mutation of the dystrophin gene, causing skeletal and cardiac muscle loss of function. The mdx mouse model of Duchenne muscular dystrophy is widely utilized to evaluate the potential of therapeutic regimens to modulate the loss of skeletal muscle function associated with dystrophin mutation. Importantly, progressive loss of diaphragm function is the most consistent striated muscle effect observed in the mdx mouse model, which is the same as in patients suffering from Duchenne muscular dystrophy.

Methods: Using the mdx mouse model, we have evaluated the effect that corticotrophin releasing factor 2 receptor (CRF2R) agonist treatment has on diaphragm function, morphology and gene expression.

Results: We have observed that treatment with the potent CRF2R-selective agonist PG-873637 prevents the progressive loss of diaphragm specific force observed during aging of mdx mice. In addition, the combination of PG-873637 with glucocorticoids not only prevents the loss of diaphragm specific force over time, but also results in recovery of specific force. Pathological analysis of CRF2R agonist-treated diaphragm muscle demonstrates that treatment reduces fibrosis, immune cell infiltration, and muscle architectural disruption. Gene expression analysis of CRF2R-treated diaphragm muscle showed multiple gene expression changes including globally decreased immune cell-related gene expression, decreased extracellular matrix gene expression, increased metabolism-related gene expression, and, surprisingly, modulation of circadian rhythm gene expression.

Conclusion: Together, these data demonstrate that CRF2R activation can prevent the progressive degeneration of diaphragm muscle associated with dystrophin gene mutation.
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http://dx.doi.org/10.1186/1741-7015-5-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1936998PMC
July 2007

Effects of a CRF2R agonist and exercise on mdx and wildtype skeletal muscle.

Muscle Nerve 2007 Sep;36(3):336-41

Department of Pediatrics, Rm. 2H18, McMaster University Medical Center, 1200 Main Street W., Hamilton, Ontario L8N 3Z5, Canada.

Corticotrophin-releasing factor 2 receptor (CRF2R) agonists prevent muscle atrophy due to immobilization, denervation, and corticosteroid-induced muscle atrophy in wildtype mice. We hypothesized that a CRF2R agonist will increase skeletal muscle mass in mdx mice. Mdx (C57BL/10ScSn-Dmd(mdx)) and wildtype (C57BL/6) mice were divided into four groups: sedentary placebo, sedentary CRF2R agonist, exercised placebo, and exercised CRF2R agonist. Mice exercised on a treadmill twice weekly for 30 min (8-12 m/min, 8 weeks). Muscle and heart weights, serum creatine kinase, and gamma-glutamyltransferase activities were measured. The CRF2R agonist increased extensor digitorum longus and soleus muscle weights (P < 0.05) in wildtype and mdx mice. Sedentary mdx CRF2R and exercised mdx placebo mice had lower serum creatine kinase activity than sedentary mdx placebo mice. CRF2R-treated mice had decreased heart weights compared to placebo-treated mice. We conclude that CRF2R agonists should be further evaluated as a potential therapy for dystrophinopathies.
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http://dx.doi.org/10.1002/mus.20820DOI Listing
September 2007

ICAT-MS-MS time course analysis of atrophying mouse skeletal muscle cytosolic subproteome.

Mol Biosyst 2005 Sep 25;1(3):229-41. Epub 2005 Jul 25.

Institute for Human Movement Sciences, ETH Zurich, and Institute of Physiology, University of Zurich, Exercise Physiology, Winterthurerstrasse 190, 8057 Zurich, Switzerland.

Skeletal muscle atrophy is a process in which protein degradation exceeds protein synthesis, resulting in a decrease of the muscle's physiological cross-sectional area and mass, and is often a serious consequence of numerous health problems. We used the isotope-coded affinity tag (ICAT) labelling approach and MS-MS to protein profile cytosolic subcellular fractions from mouse tibialis anterior skeletal muscle undergoing 0, 4, 8, or 16 days of immobilisation-induced atrophy. For the validation of peptide and protein identifications statistical algorithms were applied to the sequence database search results in order to obtain consistent sensitivity/error rates for protein and peptide identifications at each immobilisation time point. In this study, we identified and quantified a large number of mouse skeletal muscle proteins. At a protein probability (P) of P> or = 0.9 (corresponding to a false positive error rate of less than 1%) 807 proteins were identified (231, 226, 217 for 4, 8, 16 days of immobilisation and 133 for the control sample, respectively), from which 51 displayed altered protein abundance with atrophy. Due to randomness of data acquisition, a full time course could be generated only for 62 proteins, most of which displayed unchanged protein abundance. In spite of this, useful information about dataset characteristics and underlying biological processes could be obtained through gene over-representation analysis. 20 gene categories-mainly but not exclusively encoded by the subset of overlapping proteins--were consistently found to be significantly (p < 0.05) over-represented in all 4 sub-datasets.
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http://dx.doi.org/10.1039/b507839cDOI Listing
September 2005

Modifications of the human urocortin 2 peptide that improve pharmacological properties.

Peptides 2006 Jul 14;27(7):1806-13. Epub 2006 Feb 14.

Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Rd., Mason, OH 45040, USA.

Recently, we demonstrated that the corticotropin releasing factor 2 receptor agonist, urocortin 2, demonstrated anti-atrophy effects in rodent skeletal muscle atrophy models. Compared to other CRF2R agonists however, the in vivo pharmacological potency of urocortin 2 is poor when it is administered by continuous subcutaneous infusion. Therefore, we attempted to modify the structure of urocortin 2 to improve in vivo efficacy when administered by subcutaneous infusion. By substituting amino acid residues in the linker region of urocortin 2 (residues 22-32), we have demonstrated improved in vivo potency without altering selectivity, probably through reduced CRFBP binding. In addition, attempts to shorten urocortin 2 generally resulted in inactive peptides, demonstrating that the 38 amino acid urocortin 2 peptide is the minimal pharmacophore.
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http://dx.doi.org/10.1016/j.peptides.2006.01.003DOI Listing
July 2006

Phosphodiesterase 4 inhibition reduces skeletal muscle atrophy.

Muscle Nerve 2005 Dec;32(6):775-81

Research Division, Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Road, Mason, Ohio 45040, USA.

Several GTP-binding protein (G-protein)-coupled receptors that signal through Galphas (GTP-binding protein alpha stimulatory) and the cyclic adenosine monophosphate (cAMP) pathway increase skeletal muscle mass. In order to further evaluate the role of the cAMP pathway in the regulation of skeletal muscle mass, we utilized inhibitors of phosphodiesterase 4 (PDE 4), the major cAMP-modifying PDE found in skeletal muscle, to modulate skeletal muscle cAMP levels. We found that PDE 4 inhibitors reduced the loss of muscle mass and force resulting from denervation and casting in rats and mice. These studies indicate that PDE 4 inhibitors may have a role in the treatment of skeletal muscle-wasting diseases.
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http://dx.doi.org/10.1002/mus.20416DOI Listing
December 2005

Sex-based differences in skeletal muscle function and morphology with short-term limb immobilization.

J Appl Physiol (1985) 2005 Sep 28;99(3):1085-92. Epub 2005 Apr 28.

Dept. of Kinesiology, McMaster Univ., Hamilton, Ontario, Canada.

The purpose of this study was to determine the effects of short-term (14-day) unilateral leg immobilization using a simple knee brace (60 degree flexion)- or crutch-mediated model on muscle function and morphology in men (M, n = 13) and women (W, n = 14). Isometric and isokinetic (concentric-slow, 0.52 rad/s and fast, 5.24 rad/s) knee extensor peak torque was determined at three time points (Pre, Day-2, and Day-14). At the same time points, magnetic resonance imaging was used to measure the cross-sectional area of the quadriceps femoris and dual-energy X-ray absorptiometry scanning was used to calculate leg lean mass. Muscle biopsies were taken from vastus lateralis at Pre and Day-14 for myosin ATPase and myosin heavy chain analysis. Women showed greater decreases (Pre vs. Day-14) compared with men in specific strength (N/cm2) for isometric [M = 3.1 +/- 13.3, W = 17.1 +/- 15.9%; P = 0.055 (mean +/- SD)] and concentric-slow (M = 4.7 +/- 11.3, W = 16.6 +/- 18.4%; P < 0.05) contractions. There were no immobilization-induced sex-specific differences in the decrease in quadriceps femoris cross-sectional area (M = 5.7 +/- 5.0, W = 5.9 +/- 5.2%) or leg lean mass (M = 3.7 +/- 4.2, W = 2.7 +/- 2.8%). There were no fiber-type transformations, and the decreases in type I (M = 4.8 +/- 5.0, W = 5.9 +/- 3.4%), IIa (M = 7.9 +/- 9.9, W = 8.8 +/- 8.0%), and IIx (M = 10.7 +/- 10.8, W = 10.8 +/- 12.1%) fiber areas were similar between sexes. These findings indicate that immobilization-induced loss of knee extensor muscle strength is greater in women compared with men despite a similar extent of atrophy at the myofiber and whole muscle levels after 14 days of unilateral leg immobilization. Furthermore, we have described an effective and safe knee immobilization method that results in reductions in quadriceps muscle strength and size.
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http://dx.doi.org/10.1152/japplphysiol.00247.2005DOI Listing
September 2005

Sauvagine analogs selective for corticotropin releasing factor 2 receptor: effect of substitutions at positions 35 and 39 on CRF2R selectivity.

Peptides 2005 May;26(5):887-91

Procter and Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Rd., Mason, OH 45040, USA.

Corticotropin releasing factor 2 receptor selective analogs of the amphibian peptide sauvagine, a member of the corticotropin releasing factor (CRF) peptide family, have therapeutic potential for the treatment of skeletal muscle atrophy. Previously, we demonstrated that [P11X12X13]Svg peptides have improved CRF2R selectivity, although not to the level of CRF2R selective hormones such as urocortin 2 and urocortin 3. Since we also demonstrated a potential for improvement in selectivity of sauvagine by modifications of residues 35 and 39, we investigated substitutions of these amino acids in selected [P11X12X13]Svg peptides. We have observed that substitution of Arg35 in sauvagine to Ala35 (the amino acid found in all CRF2R selective agonists), increased the selectivity of [P11, X12, X13]Svg analogs. In contrast, substitution of Asp39 in sauvagine to Ala39 (also the amino acid found in all CRF2R selective agonists) did not further increase the selectivity of [P11, X12, X13, A35]Svg analogs. Thus, the residues 35 along with 11, 12, and 13 in sauvagine represent important sites for improving CRF2R selectivity.
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http://dx.doi.org/10.1016/j.peptides.2004.12.013DOI Listing
May 2005

Activation of the vasoactive intestinal peptide 2 receptor modulates normal and atrophying skeletal muscle mass and force.

J Appl Physiol (1985) 2005 Feb;98(2):655-62

Research Division, Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Rd., Mason, OH 45040-9317, USA.

Of the two known vasoactive intestinal peptide receptors (VPAC1R and VPAC2R), the VPAC2R is expressed in skeletal muscle. To evaluate the function of the VPAC2R in the physiological control of skeletal muscle mass, we utilized the VPAC1R selective agonist [K15,R16,L27]VIP(1-7) GRF(8-27)-NH2 and the VPAC2R selective agonist Ro-25-1553 to treat mice and rats undergoing either nerve damage-, corticosteroid-, or disuse-induced skeletal muscle atrophy. These analyses demonstrated that activation of VPAC2R, but not VPAC1R, reduced the loss of skeletal muscle mass and force during conditions of skeletal muscle atrophy resulting from corticosteroid administration, denervation, casting-induced disuse, increased skeletal muscle mass, and force of nonatrophying muscles. These studies indicate that VPAC2R agonists may have utility for the treatment of skeletal muscle-wasting diseases.
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http://dx.doi.org/10.1152/japplphysiol.00736.2004DOI Listing
February 2005

Discovery of corticotropin releasing factor 2 receptor selective sauvagine analogues for treatment of skeletal muscle atrophy.

J Med Chem 2005 Jan;48(1):262-5

Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Road, Mason, Ohio 45040, USA.

The corticotropin release factor 2 receptor (CRF2R) has many biological activities including modulation of the stress response. Recently, we have demonstrated that CRF2R activation functions to prevent skeletal muscle wasting resulting from a variety of physiological stimuli. Thus we are interested in identifying CRF2R selective agonists with optimal pharmacological properties for use in treating muscle wasting diseases. Several CRF2R agonists are known including the frog peptide sauvagine (Svg), which display superior pharmacological properties compared to other CRF2R agonists. Unfortunately sauvagine is a nonselective CRFR agonist, thus making it of less utility due to side effects resulting from corticotropin release factor 1 receptor (CRF1R) activation. Because our initial modifications of Svg at position 11 improved CRF2R selectivity, we investigated the role of amino acids at positions 12 and 13 in Svg. We observed that phenylalanine, leucine, isoleucine, threonine, glutamine, histidine, and tyrosine at the 12th position were the strongest promoters of CRF2R selectivity whereas phenylalanine, glutamine, trytophane, tyrosine, valine, isoleucine, leucine, and 2-naphthylalanine were the preferred residues at the 13th position. Selective sauvagine peptides demonstrated improved antiatrophy effects in a mouse-casting model when compared to sauvagine itself. Thus, we demonstrate that the CRF2R selectivity can be improved by optimizing amino acids at positions 12 and 13 (all with proline at position 11) and that the selective sauvagine analogues demonstrate better in vivo efficacy than sauvagine itself.
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http://dx.doi.org/10.1021/jm049490mDOI Listing
January 2005

Corticotropin-releasing factor 2 receptor localization in skeletal muscle.

J Histochem Cytochem 2004 Jul;52(7):967-77

Research Division, Procter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Road, Mason, OH 45040-9317, USA.

Our objective in this study was to localize the corticotropin-releasing factor 2 receptor (CRF2R) in rodent and human skeletal muscle. We found CRF2R protein to be abundant in neural tissues in skeletal muscle, including large nerve fibers and bundles, neural tissue associated with mechanoreceptors, muscle spindles, and the Golgi tendon organ. CRF2R protein was also abundant in blood vessels in skeletal muscle. CRF2R protein was also observed, although with less abundance, in the endo/perimysial regions in skeletal muscle. The localization of the CRF2R to blood vessels is consistent with the CRF2R-mediated vascular phenomena observed previously, but the observation of CRF2R in neural tissue in skeletal muscle is a novel finding with an unknown function.
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http://dx.doi.org/10.1369/jhc.4A6279.2004DOI Listing
July 2004
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