Production and Biochemical Characterization of Dimeric Recombinant Gremlin-1.

Gremlin-1 is a secreted cystine-knot protein that acts as an antagonist of bone morphogenetic proteins (BMPs), and as a ligand of heparin and the vascular endothelial growth factor receptor 2 (VEGFR2), thus regulating several physiological and pathological processes, including embryonic development, tissue fibrosis and cancer. Gremlin-1 exerts all these biological activities only in its homodimeric form. Here, we propose a multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells. Ion metal affinity chromatography (IMAC) of crude supernatant followed by heparin-affinity chromatography enables obtaining a highly pure recombinant dimeric gremlin-1 protein, exhibiting both BMP antagonist and potent VEGFR2 agonist activities.

Mechanical Properties of Human Concentrated Growth Factor (CGF) Membrane and the CGF Graft with Bone Morphogenetic Protein-2 (BMP-2) onto Periosteum of the Skull of Nude Mice.

Concentrated growth factor (CGF) is 100% blood-derived, cross-linked fibrin glue with platelets and growth factors. Human CGF clot is transformed into membrane by a compression device, which has been widely used clinically. However, the mechanical properties of the CGF membranes have not been well characterized. The aims of this study were to measure the tensile strength of human CGF membrane and observe its behavior as a scaffold of BMP-2 in ectopic site over the skull. The tensile test of the full length was performed at the speed of 2mm/min. The CGF membrane (5 × 5 × 2 mm3) or the CGF/BMP-2 (1.0 µg) membrane was grafted onto the skull periosteum of nude mice (5-week-old, male), and harvested at 14 days after the graft. The appearance and size of the CGF membranes were almost same for 7 days by soaking at 4 °C in saline. The average values of the tensile strength at 0 day and 7 days were 0.24 MPa and 0.26 MPa, respectively. No significant differences of both the tensile strength and the elastic modulus were found among 0, 1, 3, and 7 days. Supra-periosteal bone induction was found at 14 days in the CGF/BMP-2, while the CGF alone did not induce bone. These results demonstrated that human CGF membrane could become a short-term, sticky fibrin scaffold for BMP-2, and might be preserved as auto-membranes for wound protection after the surgery.

Anti-Inflammatory and Analgesic Effects of TRPV1 Polypeptide Modulator APHC3 in Models of Osteo- and Rheumatoid Arthritis.

Arthritis is a widespread inflammatory disease associated with progressive articular surface degradation, ongoing pain, and hyperalgesia causing the development of functional limitations and disability. TRPV1 channel is one of the high-potential targets for the treatment of inflammatory diseases. Polypeptide APHC3 from sea anemone Heteractis crispa is a mode-selective TRPV1 antagonist that causes mild hypothermia and shows significant anti-inflammatory and analgesic activity in different models of pain. We evaluated the anti-inflammatory properties of APHC3 in models of monosodium iodoacetate (MIA)-induced osteoarthritis and complete Freund's adjuvant (CFA)-induced rheumatoid monoarthritis in comparison with commonly used non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac, ibuprofen, and meloxicam. Subcutaneous administration of APHC3 (0.1 mg/kg) significantly reversed joint swelling, disability, grip strength impairment, and thermal and mechanical hypersensitivity. The effect of APHC3 was equal to or better than that of reference NSAIDs. Protracted treatment with APHC3 decreased IL-1b concentration in synovial fluid, reduced inflammatory changes in joints, and prevented the progression of cartilage degradation. Therefore, polypeptide APHC3 has the potential to be an analgesic and anti-inflammatory substance for the alleviation of arthritis symptoms.

Sweet as honey, bitter as bile: Mitochondriotoxic peptides and other therapeutic proteins isolated from animal tissues, for dealing with mitochondrial apoptosis.

Mitochondria are subcellular organelles involved in cell metabolism and cell life-cycle. Their role in apoptosis regulation makes them an interesting target of new drugs for dealing with cancer or rare diseases. Several peptides and proteins isolated from animal and plant sources are known for their therapeutic properties and have been tested on cancer cell-lines and xenograft murine models, highlighting their ability in inducing cell-death by triggering mitochondrial apoptosis. Some of those molecules have been even approved as drugs. Conversely, many other bioactive compounds are still under investigation for their proapoptotic properties. In this review we report about a group of peptides, isolated from animal venoms, with potential therapeutic properties related to their ability in triggering mitochondrial apoptosis. This class of compounds is known with different names, such as mitochondriotoxins or mitocans.

Growth factors-based beneficial effects of platelet lysate on umbilical cord-derived stem cells and their synergistic use in osteoarthritis treatment.

Poor viability of mesenchymal stem cells (MSCs) at the transplanted site often hinders the efficacy of MSCs-based therapy. Platelet lysate (PL) contains rich amounts of growth factors, which benefits cell growth. This study aimed to explore how human PL benefits umbilical cord-derived MSCs (huc-MSCs), and whether they have synergistic potential in osteoarthritis (OA) treatment. As quality control, flow cytometry and specific staining were performed to identify huc-MSCs, and ELISA was used to quantify growth factors in PL. CCK-8 and flow cytometry assays were performed to evaluate the effects of PL on the cell viability and cell cycle progression of huc-MSCs. Wound healing and transwell assays were conducted to assess the migration of huc-MSCs. RNA sequencing, real time PCR, and Western blot assays were conducted to explore the growth factors-based mechanism of PL. The in vitro results showed that PL significantly promoted the proliferation, cell cycle, and migration of huc-MSCs by upregulating relevant genes/proteins and activating beclin1-dependent autophagy via the AMPK/mTOR signaling pathway. The main growth factors (PDGF-AA, IGF-1, TGF-ß, EGF, and FGF) contributed to the effects of PL in varying degrees. The in vivo data showed that combined PL and huc-MSCs exerted significant synergistic effect against OA. The overall study determined the beneficial effects and mechanism of PL on huc-MSCs and indicated PL as an adjuvant for huc-MSCs in treating OA. This is the first report on the growth factors-based mechanism of PL on huc-MSCs and their synergistic application. It provides novel knowledge of PL's roles and offers a promising strategy for stem cell-based OA therapy by combining PL and huc-MSCs.

The Use of Stem Cell Differentiation Stage Factors (SCDSFs) Taken from Zebrafish Embryos during Organogenesis and Their Role in Regulating the Gene Expression of Normal and Pathological (Stem) Cells.

Studies conducted on Zebrafish embryos in our laboratory have allowed for the identification of precise moments of organogenesis in which a lot of genes are switched on and off, a sign that the genome is undergoing substantial changes in gene expression. Stem cell growth and differentiation stage-factors present in different moments of organogenesis have proven to have different specific functions in gene regulation. The substances present in the first stages of cell differentiation in Zebrafish embryos have demonstrated an ability to counteract the senescence of stem cells, reducing the expression of the beta-galactosidase marker, enhancing the genes Oct-4, Sox-2, c-Myc, TERT, and the transcription of Bmi-1, which act as key telomerase-independent repressors of cell aging. The molecules present in the intermediate to late stages of cell differentiation have proven to be able to reprogram pathological human cells, such as cancer cells and those of the basal layer of the epidermis in psoriasis, which present a higher multiplication rate than normal cells. The factors present in all the stages of cell differentiation are able to counteract neurodegeneration, and to regenerate tissues: It has been possible to regenerate hair follicles in many patients with androgenetic alopecia through transdermal administration of stem cell differentiation stage factors (SCDSFs) by means of cryopass-laser.

Screening identifies small molecules that enhance the maturation of human pluripotent stem cell-derived myotubes.

Targeted differentiation of pluripotent stem (PS) cells into myotubes enables in vitro disease modeling of skeletal muscle diseases. Although various protocols achieve myogenic differentiation in vitro, resulting myotubes typically display an embryonic identity. This is a major hurdle for accurately recapitulating disease phenotypes in vitro, as disease commonly manifests at later stages of development. To address this problem, we identified four factors from a small molecule screen whose combinatorial treatment resulted in myotubes with enhanced maturation, as shown by the expression profile of myosin heavy chain isoforms, as well as the upregulation of genes related with muscle contractile function. These molecular changes were confirmed by global chromatin accessibility and transcriptome studies. Importantly, we also observed this maturation in three-dimensional muscle constructs, which displayed improved in vitro contractile force generation in response to electrical stimulus. Thus, we established a model for in vitro muscle maturation from PS cells.

Concentrated growth factor promotes proliferation, osteogenic differentiation, and angiogenic potential of rabbit periosteum-derived cells in vitro.

OBJECTIVE: The aim of this research is to investigate the effects of concentrated growth factor (CGF) on the proliferation, osteogenic differentiation, and angiogenic potential of rabbit periosteum-derived cells (PDCs) in vitro. METHODS: PDCs were isolated from the femoral and tibial periosteum of rabbits and cultured with or without CGF membranes or CGF conditioned media. Scanning electron microscopy (SEM) was used for the structural characterization. Cell Counting Kit-8 assay was used to measure cell proliferation. Alkaline phosphatase (ALP) activity of PDCs was also measured. Immunohistochemistry was used to detect the expression of CD34. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time PCR (qPCR), and Western blot were used to evaluate the secretion and expression levels of osteogenic differentiation markers (bone morphogenetic protein-2, type I collagen, osteocalcin) and angiogenesis markers (vascular endothelial growth factor, basic fibroblast growth factor) in supernatants and PDCs at days 3, 7, 14, and 21. RESULTS: The SEM analysis showed a dense three-dimensional fibrin network in CGF, and CGF membranes were covered by PDCs with elongated or polygonal morphological features. Compared with the control group, CGF significantly promoted the proliferation of PDCs during the experimental period (p < 0.05). Immunohistochemistry revealed that PDCs were dispersedly distributed among the CGF substrates, and CD34-positive cells were also present. Moreover, CGF significantly increased the ALP activity and upregulated the expression and secretion of osteogenic differentiation and angiogenesis markers in PDCs at days 3, 7, 14, and 21 (p < 0.05). CONCLUSION: CGF can increase the proliferation and promote the osteogenic differentiation and angiogenic potential of PDCs in vitro. These results indicate that CGF can be used as a new therapeutic means for biotechnological and clinical applications.

The Fate of Molecular Oxygen in Azinomycin Biosynthesis.

The azinomycins are a family of aziridine-containing antitumor antibiotics and represent a treasure trove of biosynthetic reactions. The formation of the azabicyclo[3.1.0]hexane ring and functionalization of this ring system remain the least understood aspects of the pathway. This study reports the incorporation of 18O-labeled molecular oxygen in azinomycin biosynthesis including both oxygens of the diol that ultimately adorn the aziridino[1,2- a]pyrrolidine moiety. Likewise, two other sites of heavy atom incorporation are observed.

Concentrated growth factor exudate enhances the proliferation of human periodontal ligament cells in the presence of TNF­α.

The purpose of this study was to evaluate the effects of concentrated growth factor exudate (CGFe) on human periodontal ligament cells (hPDLCs) stimulated by tumor necrosis factor (TNF)­α. From the peripheral blood of healthy donors, CGFe was prepared according to the Sacco protocol after 7 days of incubation. The hPDLCs were cultured by a tissue explant method and identified with anti­vimentin and anti­cytokeratin antibodies. Cells were subjected to four different treatments: i) Control; ii) TNF­α (10 ng/ml); iii) CGFe (concentration 50%); and iv) CGFe+TNF­α. The proliferation of hPDLCs was measured with Cell Counting Kit­8 assays. Osteogenic differentiation and mineralization were determined by Alizarin Red S staining, alkaline phosphatase activity, western blotting and reverse transcription­quantitative polymerase chain reaction. CGFe enhanced cell proliferation and upregulated ALP activity, the mineralization level, and osteogenic­associated osteocalcin, runt­related transcription factor 2 and Osterix gene expression in hPDLCs under inflammatory conditions induced by TNF­α. The present study demonstrated that CGFe enhanced hPDLC proliferation and osteogenic differentiation in the presence of TNF­α­induced inflammation. As CGFe can be obtained from the venous blood of patients, it generates no immune reaction. Thus, it is more economical and beneficial to use CGFe in clinical periodontal regeneration practice than synthetic growth factors.

High-throughput combinatorial screening reveals interactions between signaling molecules that regulate adult neural stem cell fate.

Advancing our knowledge of how neural stem cell (NSC) behavior in the adult hippocampus is regulated has implications for elucidating basic mechanisms of learning and memory as well as for neurodegenerative disease therapy. To date, numerous biochemical cues from the endogenous hippocampal NSC niche have been identified as modulators of NSC quiescence, proliferation, and differentiation; however, the complex repertoire of signaling factors within stem cell niches raises the question of how cues act in combination with one another to influence NSC physiology. To help overcome experimental bottlenecks in studying this question, we adapted a high-throughput microculture system, with over 500 distinct microenvironments, to conduct a systematic combinatorial screen of key signaling cues and collect high-content phenotype data on endpoint NSC populations. This novel application of the platform consumed only 0.2% of reagent volumes used in conventional 96-well plates, and resulted in the discovery of numerous statistically significant interactions among key endogenous signals. Antagonistic relationships between fibroblast growth factor 2, transforming growth factor ß (TGF-ß), and Wnt-3a were found to impact NSC proliferation and differentiation, whereas a synergistic relationship between Wnt-3a and Ephrin-B2 on neuronal differentiation and maturation was found. Furthermore, TGF-ß and bone morphogenetic protein 4 combined with Wnt-3a and Ephrin-B2 resulted in a coordinated effect on neuronal differentiation and maturation. Overall, this study offers candidates for further elucidation of significant mechanisms guiding NSC fate choice and contributes strategies for enhancing control over stem cell-based therapies for neurodegenerative diseases.

Nrf2 activation and down-regulation of HMGB1 and MyD88 expression by amnion membrane extracts in response to the hypoxia-induced injury in cardiac H9c2 cells.

BACKGROUND: human Amniotic Membrane (hAM) extracts contain bioactive molecules such as growth factors and cytokines. Studies have confirmed the ability of hAM in reduction of post-operative dysfunction in patients with cardiac surgery. However, the function of Amniotic Membrane Proteins (AMPs), extracted from hAM, against hypoxia-induced H9c2 cells injury have never been investigated. In this study, we aimed to appraise the protective impact of AMPs on H9c2 cells under hypoxia condition. METHODS: Cardiomyocyte cells were pre-incubated with AMPs and subjected to 24 h hypoxia to elucidate its effects on expression of Nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1(HO-1). Furthermore, the high mobility group box-1 (HMGB1) and Myeloid differentiation primary response 88 (MyD88) expressions were detected by qPCR and western-blotting. The mitochondrial membrane potential (ΔΨm) was estimated by JC-1 using fluorescent microscopy and fluorimetry. Moreover, the cell apoptosis and intracellular calcium levels were measured by flow cytometry. RESULTS: Pre-treatment of AMPs resulted in significant induction in cell viability and decreased the LDH release under hypoxic condition in H9c2 cells. Accordingly, these protective effects of AMPs were associated with a reduction in apoptosis rates and intracellular Ca2+, meanwhile, ΔΨm was increased. Pre-treatment with AMPs resulted in degradation of HMGB1 and MyD88 levels and depicted pro-survival efficacy of AMPs against hypoxia-induced cell damage through induction of HO-1 and Nrf2. CONCLUSION: The data indicated that AMPs mediated HO-1 regulation by Nrf2 activation and plays critical protective effects in hypoxia-induced H9c2 injury in vitro by the inhibition of myocardial HMGB1 and MyD88 inflammatory cascade.

Alanine mother liquor as a nitrogen source for docosahexaenoic acid production by Schizochytrium sp. B4D1

Alanine mother liquor, a type of industrial waste from alanine fermentation, was used as a nitrogen source to produce docosahexaenoic acid (DHA) by Schizochytrium sp. B4D1. The results indicated that yeast extract could trigger the utilization of the alanine mother liquor. Additionally, the alanine can be quenched during the culture, which aids in DHA accumulation. The medium components were optimized via response surface methodology as follows: 99.98-g/L glucose, 0.05-g/L yeast extract and a 183.17 dilution factor of the alanine mother liquid (v/v, with an alanine content of 0.72 g/L) and 17.98% inoculum concentration (v/v). Finally, in a 50-mL shake-flask fermentation, the DHA yield was 2.29 g/L.

Pro-inflammatory cytokines and growth factors in human milk: an exploratory analysis of racial differences to inform breast cancer etiology.

BACKGROUND: Analysis of cytokines and growth factors in human milk offers a noninvasive approach for studying the microenvironment of the postpartum breast, which may better reflect tissue levels than testing blood samples. Given that Black women have a higher incidence of early-onset breast cancers than White women, we hypothesized that milk of the former contains higher levels of pro-inflammatory cytokines, adipokines, and growth factors. METHODS: Participants included 130 Black and 162 White women without a history of a breast biopsy who completed a health assessment questionnaire and donated milk for research. Concentrations of 15 analytes in milk were examined using two multiplex and 4 single-analyte electrochemiluminescent sandwich assays to measure pro-inflammatory cytokines, angiogenesis factors, and adipokines. Mixed-effects ordinal logistic regression was used to identify determinants of analyte levels and to compare results by race, with adjustment for confounders. Factor analysis was used to examine covariation among analytes. RESULTS: Thirteen of 15 analytes were detected in ≥ 25% of the human milk specimens. In multivariable models, elevated BMI was significantly associated with increased concentrations of 5 cytokines: IL-1ß, bFGF, FASL, EGF, and leptin (all p-trend < 0.05). Black women had significantly higher levels of leptin and IL-1ß, controlling for BMI. Factor analysis of analyte levels identified two factors related to inflammation and growth factor pathways. CONCLUSION: This exploratory study demonstrated the feasibility of measuring pro-inflammatory cytokines, adipokines, and angiogenesis factors in human milk, and revealed higher levels of some pro-inflammatory factors, as well as increased leptin levels, among Black as compared with White women.

The logic of biologically active small molecules: amazing ability of microorganisms.

In this review article, I will outline my way of thinking about biologically active small molecules. The structure of liposidomycin B from Streptomyces species resulted in my initial sense that a structure tells its function. A biologically active small molecule may save directly or indirectly a number of people. Even if the molecule has not been used as a therapeutic agent, it can be used as a useful chemical probe for dissecting a living cell into different biochemical pieces. Such biologically active small molecules derived from microorganisms have been primarily found in cultivable microorganisms that make up only 1% of total microbes in nature. Discovery of novel growth factors, zincmethylphyrin, zinc coproporphyrin, and coproporphyrin enabled laboratory cultivation of previously uncultured Leucobacter sp. These findings might expand the possibility for further discovery of novel therapeutic agents or chemical probes.

Exosomes derived from human umbilical cord blood mesenchymal stem cells stimulates rejuvenation of human skin.

Human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) play an important role in cutaneous wound healing, and recent studies suggested that MSC-derived exosomes activate several signaling pathways, which are conducive in wound healing and cell growth. In this study, we investigated the roles of exosomes that are derived from USC-CM (USC-CM Exos) in cutaneous collagen synthesis and permeation. We found that USC-CM has various growth factors associated with skin rejuvenation. Our in vitro results showed that USC-CM Exos integrate in Human Dermal Fibroblasts (HDFs) and consequently promote cell migration and collagen synthesis of HDFs. Moreover, we evaluated skin permeation of USC-CM Exos by using human skin tissues. Results showed that Exo-Green labeled USC-CM Exos approached the outermost layer of the epidermis after 3 h and gradually approached the epidermis after 18 h. Moreover, increased expressions of Collagen I and Elastin were found after 3 days of treatment on human skin. The results showed that USC-CM Exos is absorbed into human skin, it promotes Collagen I and Elastin synthesis in the skin, which are essential to skin rejuvenation and shows the potential of USC-CM integration with the cosmetics or therapeutics.

Differential effects of amnion and chorion membrane extracts on osteoblast-like cells due to the different growth factor composition of the extracts.

Human amniotic membrane extracts contain numerous growth factors and bioactive substances. However, osteogenic effects of amnion and chorion membrane extracts (AME and CME, respectively) on osteoblasts are unclear. In this study, we explored the ability of AME and CME to promote the osteogenic differentiation of osteoblast-like MG-63 cells. MG-63 cells were cultured in osteogenic induction medium (OIM) with or without exogenous AME and CME. CME enhanced the osteogenic differentiation of MG-63 cells compared with AME, as indicated by increased mineralization; alkaline phosphatase activity; and mRNA expression of osteogenic marker genes encoding integrin-binding sialoprotein (IBSP), RUNX2, OSTERIX, and osteocalcin (OCN). Interestingly, AME and CME contained different combinations of osteogenesis-related growth factors, including basic fibroblast growth factor (bFGF), transforming growth factor beta-1 (TGFß-1), and epidermal growth factor (EGF), which differentially regulated the osteogenic differentiation of MG-63 cells. bFGF and TGFß-1 present in CME positively regulated the osteogenic differentiation of MG-63 cells, whereas EGF present in AME negatively regulated the differentiation of MG-63 cells. Moreover, exogenous treatment of EGF antagonized CME-induced mineralization of extracellular matrix on MG-63 cells. We compared the osteogenic efficacy of CME with that of BMP2, bFGF, and TGFß-1 alone or their combinations. We observed that CME greatly enhanced osteogenesis by providing a conductive environment for the differentiation of MG-63 cells. Together, our results indicated that human AME and CME exerted differential effects on osteogenesis because of the presence of different compositions of growth factors. In addition, our results highlighted a new possible strategy of using CME as a biocompatible therapeutic material for bone regeneration.

Long-term cytokine and growth factor release from equine platelet-rich fibrin clots obtained with two different centrifugation protocols.

OBJECTIVES: To compare the temporal release (over three weeks) of tumor necrosis factor alpha (TNF-α), interleukin 4 (IL-4), IL-1 receptor antagonist (IL-1ra), platelet-derived growth factor BB (PDGF-BB) and transforming growth factor beta-1 (TGF-ß1) from two platelet-rich fibrin (PRF) preparations from equine blood obtained at either 240g/8min or 416g/10min. METHODS: Whole blood from 10 horses was used to obtain PRF clots by two different centrifugation protocols. After 1h of rest, PRF clots were deposited in wells with culture medium, which was changed at 6h, 24h and then every 48h to 21days. Cytokines and GFs were measured by ELISA at 1h (serum supernatants from PRF clots) and all time points of culture medium change. A negative control (plasma) and a positive control (blood lysate) were also included. RESULTS: There were no relevant differences between the two protocols for the temporal release of proteins. However, a significant (p=0.01) effect of time was noted. All cytokines were detected after 6h of PRF clot culture until day 21. GF were detected at 1h until day 21. The concentrations for these proteins diminished gradually over time. A highly significant (p=0.01) correlation was noticed between all the proteins evaluated. CONCLUSIONS: Leukocytes enmeshed in PRF clots were able to produce cytokines, TGF-ß1 and PDGF-BB. These findings demonstrate a paramount role of leukocytes in wound healing induced or modified by PRF clots in mammals.

Engineering potent long-acting variants of the Wnt inhibitor DKK2.

Wnt signaling pathways are required for a wide variety of biological processes ranging from embryonic development to tissue repair and regeneration. Dickkopf-2 (DKK2) is classically defined as a canonical Wnt inhibitor, though it may play a role in activating non-canonical Wnt pathways in the context of endothelial network formation after acute injury. Here we report the discovery of a fusion partner for a DKK2 polypeptide that significantly improves the expression, biochemical properties and pharmacokinetics (PK) of the DKK2 polypeptide. Specifically, human serum albumin (HSA) was identified as a highly effective fusion partner. Substitution of selected amino acid residues in DKK2 designed to decrease heparan sulfate binding by HSA-DKK2 variants, further improved the PK properties of the molecule in rodents. The HSA-DKK2 variants were monomeric, as thermally stable as wild type, and active as measured by their ability to bind to and prevent phosphorylation of the Wnt coreceptor LRP6. Our engineering efforts resulted in potent long-lived variants of the canonical Wnt inhibitor DKK2, applicable for Wnt pathway manipulation either by systematic delivery or focused administration at sites of tissue injury.

The Anti-Aging Effects of Low Oxygen Tension Generated Multipotent Growth Factor Containing Serum.

Growth factors are a new category of ingredient found in modern cosmeceutical formulations. One novel method of obtaining cosmeceutical growth factors is the use of a bioreactor to culture neonatal broblasts on dextran microcarrier beads for 8 weeks under low oxygen tension (1-5%) mimicking embryonic conditions and eliminating the need for fetal bovine serum constituents in the final cosmetic material. This research evaluated the ingredient in a moisturizing vehicle on 40 females to determine its efficacy in improving overall facial skin appearance, as well as skin brightness, evenness, firmness, pore size, radiance, fine lines, coarse wrinkles, and blotchiness/ dispigmentation. Statistically significant improvement was seen in 90 days in skin hydration through corneometry, as well in global investigator and subject assessments. J Drugs Dermatol. 2017;16(1):30-34..