Publications by authors named "Michael J Flagler"

10 Publications

  • Page 1 of 1

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

Int J Cosmet Sci 2021 Oct 25;43(5):518-529. Epub 2021 Aug 25.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/ics.12725DOI Listing
October 2021

Reductive Stress Selectively Disrupts Collagen Homeostasis and Modifies Growth Factor-independent Signaling Through the MAPK/Akt Pathway in Human Dermal Fibroblasts.

Mol Cell Proteomics 2019 06 19;18(6):1123-1137. Epub 2019 Mar 19.

From the ‡The Department of Biosciences, Durham University, Stockton Road, Durham, DH1 3LE, UK;

Redox stress is a well-known contributor to aging and diseases in skin. Reductants such as dithiothreitol (DTT) can trigger a stress response by disrupting disulfide bonds. However, the quantitative response of the cellular proteome to reductants has not been explored, particularly in cells such as fibroblasts that produce extracellular matrix proteins. Here, we have used a robust, unbiased, label-free SWATH-MS proteomic approach to quantitate the response of skin fibroblast cells to DTT in the presence or absence of the growth factor PDGF. Of the 4487 proteins identified, only 42 proteins showed a statistically significant change of 2-fold or more with reductive stress. Our proteomics data show that reductive stress results in the loss of a small subset of reductant-sensitive proteins (including the collagens COL1A1/2 and COL3A1, and the myopathy-associated collagens COL6A1/2/3), and the down-regulation of targets downstream of the MAPK pathway. We show that a reducing environment alters signaling through the PDGF-associated MAPK/Akt pathways, inducing chronic dephosphorylation of ERK1/2 at Thr202/Tyr204 and phosphorylation of Akt at Ser473 in a growth factor-independent manner. Our data highlights collagens as sentinel molecules for redox stress downstream of MAPK/Akt, and identifies intervention points to modulate the redox environment to target skin diseases and conditions associated with erroneous matrix deposition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/mcp.RA118.001140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6553930PMC
June 2019

Incubatory environment of the scalp impacts pre-emergent hair to affect post-emergent hair cuticle integrity.

J Cosmet Dermatol 2018 Feb 14;17(1):105-111. Epub 2017 May 14.

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

Objectives: To determine whether the oxidative stress transmitted to newly grown hair from an unhealthy scalp has physical consequences to the cuticular condition and function.

Methods: A uniquely designed 24-week clinical study included 8 weeks of pretreatment with a cosmetic shampoo and 16 weeks of treatment with either a potentiated zinc pyrithione (ZPT) antidandruff shampoo or a placebo cosmetic shampoo. This clinical design allowed the growth and acquisition of hair samples under conditions of varying but known scalp health as a result of treating a dandruff/seborrheic dermatitis (D/SD) population. Two complementary methods were used to characterize the integrity of the cuticular surface. Hair surface hydrophobicity was assessed by quantifying water wetting force using a Wilhelmy balance method. Surface structure and porosity were assessed using dynamic vapor sorption (DVS) to gravimetrically quantify water sorption.

Results: Chemical oxidative stress to pre-emergent hair has been shown to have negative consequences to hair surface structure. Compared to a placebo shampoo control, use of a potentiated ZPT shampoo improved scalp health and significantly improved the following attributes associated with healthy hair: hair surface hydrophobicity (surface energy) and cuticular moisture barrier effectiveness (dynamic vapor sorption).

Conclusions: Pre-emergent hair can be negatively impacted by the oxidative stress that occurs with an unhealthy scalp, possibly due to metabolic activity of resident microbes. Manifestations of the oxidative stress include altered cuticle surface properties that are responsible for its protective function; these effects are similar in type to those observed by bleaching post-emergent hair. These alterations have the potential to make the hair, once emerged from the scalp, more susceptible to the cumulative physical and chemical insults responsible for hair feel and look, fiber integrity, and overall retention.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/jocd.12355DOI Listing
February 2018

Human hair shaft proteomic profiling: individual differences, site specificity and cuticle analysis.

PeerJ 2014 5;2:e506. Epub 2014 Aug 5.

Forensic Science Graduate Program and Department of Environmental Toxicology, University of California , Davis, CA , USA.

Hair from different individuals can be distinguished by physical properties. Although some data exist on other species, examination of the individual molecular differences within the human hair shaft has not been thoroughly investigated. Shotgun proteomic analysis revealed considerable variation in profile among samples from Caucasian, African-American, Kenyan and Korean subjects. Within these ethnic groups, prominent keratin proteins served to distinguish individual profiles. Differences between ethnic groups, less marked, relied to a large extent on levels of keratin associated proteins. In samples from Caucasian subjects, hair shafts from axillary, beard, pubic and scalp regions exhibited distinguishable profiles, with the last being most different from the others. Finally, the profile of isolated hair cuticle cells was distinguished from that of total hair shaft by levels of more than 20 proteins, the majority of which were prominent keratins. The cuticle also exhibited relatively high levels of epidermal transglutaminase (TGM3), accounting for its observed low degree of protein extraction by denaturants. In addition to providing insight into hair structure, present findings may lead to improvements in differentiating hair from various ethnic origins and offer an approach to extending use of hair in crime scene evidence for distinguishing among individuals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7717/peerj.506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4137660PMC
August 2014

Molecular basis of differential B-pentamer stability of Shiga toxins 1 and 2.

PLoS One 2010 Dec 28;5(12):e15153. Epub 2010 Dec 28.

Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America.

Escherichia coli strain O157:H7 is a major cause of food poisoning that can result in severe diarrhea and, in some cases, renal failure. The pathogenesis of E. coli O157:H7 is in large part due to the production of Shiga toxin (Stx), an AB(5) toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. There are two major isoforms, Stx1 and Stx2, which differ dramatically in potency despite having 57% sequence identity. Animal studies and epidemiological studies show Stx2 is associated with more severe disease. Although the molecular basis of this difference is unknown, data suggest it is associated with the B-subunit. Mass spectrometry studies have suggested differential B-pentamer stability between Stx1 and Stx2. We have examined the relative stability of the B-pentamers in solution. Analytical ultracentrifugation using purified B-subunits demonstrates that Stx2B, the more deadly isoform, shows decreased pentamer stability compared to Stx1B (EC(50) = 2.3 µM vs. EC(50) = 0.043 µM for Stx1B). X-ray crystal structures of Stx1B and Stx2B identified a glutamine in Stx2 (versus leucine in Stx1) within the otherwise strongly hydrophobic interface between B-subunits. Interchanging these residues switches the stability phenotype of the B-pentamers of Stx1 and Stx2, as demonstrated by analytical ultracentrifugation and circular dichroism. These studies demonstrate a profound difference in stability of the B-pentamers in Stx1 and Stx2, illustrate the mechanistic basis for this differential stability, and provide novel reagents to test the basis for differential pathogenicity of these toxins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0015153PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3010993PMC
December 2010

Shiga toxin subtypes display dramatic differences in potency.

Infect Immun 2011 Mar 3;79(3):1329-37. Epub 2011 Jan 3.

Molecular Genetics, Biochemistry, and Microbiology, Room 3109, 231 Albert Sabin Way, ML 524, University of Cincinnati, Cincinnati, OH 45267-0524, USA.

Purified Shiga toxin (Stx) alone is capable of producing systemic complications, including hemolytic-uremic syndrome (HUS), in animal models of disease. Stx includes two major antigenic forms (Stx1 and Stx2), with minor variants of Stx2 (Stx2a to -h). Stx2a is more potent than Stx1. Epidemiologic studies suggest that Stx2 subtypes also differ in potency, but these differences have not been well documented for purified toxin. The relative potencies of five purified Stx2 subtypes, Stx2a, Stx2b, Stx2c, Stx2d, and activated (elastase-cleaved) Stx2d, were studied in vitro by examining protein synthesis inhibition using Vero monkey kidney cells and inhibition of metabolic activity (reduction of resazurin to fluorescent resorufin) using primary human renal proximal tubule epithelial cells (RPTECs). In both RPTECs and Vero cells, Stx2a, Stx2d, and elastase-cleaved Stx2d were at least 25 times more potent than Stx2b and Stx2c. In vivo potency in mice was also assessed. Stx2b and Stx2c had potencies similar to that of Stx1, while Stx2a, Stx2d, and elastase-cleaved Stx2d were 40 to 400 times more potent than Stx1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/IAI.01182-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067513PMC
March 2011

Comparison of binding platforms yields insights into receptor binding differences between shiga toxins 1 and 2.

Biochemistry 2010 Mar;49(8):1649-57

Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio 45267, USA.

Protein-glycan interactions are typically very weak, and avid binding is achieved when proteins express multiple glycan binding sites. Shiga toxin (Stx) uses glycan receptors to enter cells. Stx has five identical binding subunits, each with three nonidentical glycan binding sites. Previous studies examined binding to biantennary glycans expressing Pk trisaccharide mimics immobilized on streptavidin, resulting in display of four trisaccharides per streptavidin face. Stx1 preferred the Pk trisaccharide of its native receptor, globotriaosylceramide (Gb3), while the more potent and clinically relevant variant, Stx2, preferred the Pk trisaccharide with the terminal galactose replaced with N-acetylgalactosamine (NHAc-Pk). In the present study, binding of Stxs to Pk analogues was examined using two experimental platforms, ELISA and surface plasmon resonance (SPR). ELISA was more sensitive than SPR. Sensitivity in the ELISA was due to high streptavidin density, suggesting that avid binding may require engagement of more than four trisaccharides. Selectivity for the Pk analogues was maintained in both experimental platforms. Glycan preference was mapped to binding site 2, since reciprocal mutation of a single amino acid (asparagine 32 of Stx1 B-subunit/serine 31 of Stx2 B-subunit) reversed binding preference. However, native Stx1 bound well to plates loaded with a 50:50 mixture of Pk-NHAc-Pk, while Stx2 bound less efficiently, suggesting that one of the Stx1 binding sites may only engage Pk, while another may tolerate either Pk or NHAc-Pk. Varying glycan structure and density across different in vitro binding platforms revealed important differences in receptor binding properties between Stx1 and Stx2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/bi902084yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2857392PMC
March 2010

Characterization of a two-component regulatory system from Acinetobacter baumannii that controls biofilm formation and cellular morphology.

Microbiology (Reading) 2008 Nov;154(Pt 11):3398-3409

Department of Microbiology, Miami University, Oxford, OH 45056, USA.

Acinetobacter baumannii forms biofilms on abiotic surfaces, a phenotype that may explain its ability to survive in nosocomial environments and to cause device-related infections in compromised patients. The biofilm proficiency of the 19606 type strain depends on the production of pili, cell-surface appendages assembled via the CsuAB-A-B-C-D-E chaperone-usher secretion system. The screening of a bank of isogenic insertion derivatives led to the identification of a biofilm-deficient derivative in which a transposon insertion disrupted a gene predicted to encode the response regulator of a two-component regulatory system. This gene, which was named bfmR, is required for the expression of the Csu pili chaperone-usher assembly system. This coding region is followed by an ORF encoding a putative sensor kinase that was named bfmS, which plays a less relevant role in biofilm formation when cells are cultured in rich medium. Further examination showed that the bfmR mutant was capable of attaching to abiotic surfaces, although to levels significantly lower than those of the parental strain, when it was cultured in a chemically defined minimal medium. Additionally, the morphology of planktonic cells of this mutant, when grown in minimal medium, was drastically affected, while adherent mutant cells were indistinguishable in shape and size from the parental strain. Together, these results indicate that BfmR is part of a two-component regulatory system that plays an important role in the morphology of A. baumannii 19606 cells and their ability to form biofilms on abiotic surfaces.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/mic.0.2008/019471-0DOI Listing
November 2008

Comparative analysis of the abilities of Shiga toxins 1 and 2 to bind to and influence neutrophil apoptosis.

Infect Immun 2007 Feb 13;75(2):760-5. Epub 2006 Nov 13.

Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, OH 45267, USA.

Hemolytic-uremic syndrome (HUS), the life-threatening complication following infection by the intestinal pathogen Escherichia coli O157:H7, is due to the ability of the pathogen to produce toxins in the Shiga toxin (Stx) family. Activated neutrophils are observed in HUS patients, yet it is unclear whether Stx exerts a direct effect on neutrophils or whether the toxin acts indirectly. The effect of Stx1 and Stx2 on human neutrophils was examined. Neither Stx1 nor Stx2 altered the rate of neutrophil apoptosis. Minimal binding of either toxin to neutrophils was observed, and the toxin was easily eluted from the cells. Stx1 and Stx2 were found to circulate in the plasma of mice following intravenous injection, and both toxins were cleared rapidly from the blood. Together these results suggest that neither Stx1 nor Stx2 interacts directly with neutrophils.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/IAI.01594-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1828505PMC
February 2007
-->