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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.

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 mm 3 ) 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.

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.

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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-β, 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.

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.

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., Competing Interests: SS, RM, BX, AM, HK, JL, JK, HM, NT, WS, RP No competing interests declared, FR, JA is affiliated with Bio-Techne. The author has no financial interests to declare., (© 2019, Selvaraj et al.)

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 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 18 O-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.

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 anti‑vimentin and anti‑cytokeratin antibodies. Cells were subjected to four different treatments: i) Control; ii) TNF‑α (10 ng/ml); iii) CGFe (concentration 50%); and iv) CGFe+TNF‑α. The proliferation of hPDLCs was measured with Cell Counting Kit‑8 assays. Osteogenic differentiation and mineralization were determined by Alizarin Red S staining, alkaline phosphatase activity, western blotting and reverse transcription‑quantitative polymerase chain reaction. CGFe enhanced cell proliferation and upregulated ALP activity, the mineralization level, and osteogenic‑associated osteocalcin, runt‑related transcription factor 2 and Osterix gene expression in hPDLCs under inflammatory conditions induced by TNF‑α. The present study demonstrated that CGFe enhanced hPDLC proliferation and osteogenic differentiation in the presence of TNF‑α‑induced 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.

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 β (TGF-β), 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-β 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., (© 2018 Wiley Periodicals, Inc.)

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., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

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-1β, bFGF, FASL, EGF, and leptin (all p-trend < 0.05). Black women had significantly higher levels of leptin and IL-1β, 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.

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.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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-β 1 ) 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-β 1 and PDGF-BB. These findings demonstrate a paramount role of leukocytes in wound healing induced or modified by PRF clots in mammals., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 (TGFβ-1), and epidermal growth factor (EGF), which differentially regulated the osteogenic differentiation of MG-63 cells. bFGF and TGFβ-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 TGFβ-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.

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 Author 2017. Published by Oxford University Press.)

Concentrated growth factors (CGFs), the new generation of platelet concentrate products, appear to exhibit superior potential for tissue regeneration. However, there are only a few studies supporting this. This study was designed to investigate the effect of CGFs on proliferation and alkaline phosphatase (ALP) activity of human periodontal ligament cells (hPDLCs) in vitro. Furthermore, as bone homeostasis is fundamentally controlled by Wnt-mediated signals, we also investigated Wnt3a expression of hPDLCs after treatment of CGFs. hPDLCs and CGFs were obtained from the same volunteer. CGFs or combination of recombined human TGF-β1 (rhTGF-β1) and PDGF-AB (rhPDGF-AB) were added to hPDLCs in different concentrations. The rate of proliferation was analyzed by an MTT assay. ALP activity was assessed using p-NPP assay. Quantitative RT-PCR was used to evaluate the gene expression of Wnt3a. In a range of concentrations, CGFs significantly promoted the proliferation of hPDLCs in a dose-dependent manner. ALP activity was also enhanced by CGFs in a dose-dependent and time-dependent manner. The stimulatory effect of CGFs was much greater than rhTGF-β1 and rhPDGF-AB combination. Quantitative RT-PCR results showed that Wnt3a mRNA expression was increased at 24 h in hPDLCs treated by CGFs. CGFs can enhance hPDLCs proliferation and ALP activity and may have great potential in clinical and biotechnological applications.

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..

Platelet concentrates, such as Concentrated Growth Factors (CGF), are autologous preparations obtained from the patient's own blood and rich in platelets, growth factors and cytokines involved in the key processes of tissue regeneration. These autologous concentrates differ in the way of preparation and also in the content of platelets, growth factors and leucocytes, as well as in the fibrin network architecture. So it is difficult to have a standardized product. The aim of the present study was to evaluate how the use of test tubes of different material, for blood collection, could influence the CGF production. Three different test tubes were used and the obtained CGFs were subjected to histomorphological and immunohistochemical analyses. Results showed that the tube material and shape influenced the CGF composition. In fact, according to the type of tube used, the obtained CGFs showed differences in morphology, in the fibrin network architecture and in blood cell localization and distribution., (© 2016 Wiley Periodicals, Inc.)

CS5931 is a novel anticancer agent isolated from the sea squirt Ciona savignyi. However, its content in the species is very low, and developing a novel approach for production of the polypeptide is promising. In the present study, we expressed and purified the polypeptide from E. coli, and the fermentation conditions were studied using response surface methodology. The yield of CS5931 was increased from 2.0 to 7.5 mg/L. The denaturing and renaturation conditions were also studied. Using the optimized renaturation condition, the anticancer activity of refolding CS5931 was increased significantly; the value of IC50 was decreased from 23.2 to 11.6 μM. In vivo study using xenograft nude mice bearing HCT116 cancer cells revealed that CS5931 was able to inhibit the growth of tumor significantly. The study provides a useful approach for obtaining enough amount of CS5931 for further study. This study is also important for developing the polypeptide as a novel anticancer agent.

We have identified coproporphyrins including structurally new zincmethylphyrins I and III as growth factors A-F for the previously uncultured bacterial strain, Leucobacter sp. ASN212, from a supernatant of 210 l of Sphingopyxis sp. GF9 culture. Growth factors A-F induced significant growth of strain ASN212 at the concentrations of picomolar to nanomolar which would otherwise be unculturable in liquid medium or on agar plate. More interestingly, we found that the growth factors functioned as self-toxic compounds for the growth-factor producing strain GF9 at the picomolar to nanomolar levels. As a variety of bacteria could potentially produce coproporphyrins, our findings suggest that these compounds function as a novel class of signal molecules across a boundary at phylum level in the complex bacterial communities.

Murine herpesvirus 68 (MHV-68) can transform cells in vitro and in vivo. We investigated putative murine herpesvirus growth factors (MHGFs) obtained by the separation of cell-free media from MHV-68-transformed cells on an FPLC Sephadex G15 column. The transforming activity of the MHGFA fraction was related to depolymerization of actin, disruption of the microtubule network, and punctate-reticular changes of the Golgi. The MHGFW fraction had only repressing activity on the transformed phenotype. Incubation of MRC-5 cells with MHGFW resulted in reticular changes of the Golgi apparatus, minor depolymerization of actin filaments, and no detectable changes of the microtubule network. Reorganization of the actin cytoskeleton is associated with oncogenesis. Further study of the MHGFs from herpesviruses and proteins responsible for changes in the organization of the cytoskeleton could give insight into the cell transformation mechanism and oncogenesis., (© 2017 S. Karger AG, Basel.)

Skeletal muscle has emerged as an important secretory organ that produces so-called myokines, regulating energy metabolism via autocrine, paracrine, and endocrine actions; however, the nature and extent of the muscle secretome has not been fully elucidated. Mass spectrometry (MS)-based proteomics, in principle, allows an unbiased and comprehensive analysis of cellular secretomes; however, the distinction of bona fide secreted proteins from proteins released upon lysis of a small fraction of dying cells remains challenging. Here we applied highly sensitive MS and streamlined bioinformatics to analyze the secretome of lipid-induced insulin-resistant skeletal muscle cells. Our workflow identified 1073 putative secreted proteins including 32 growth factors, 25 cytokines, and 29 metalloproteinases. In addition to previously reported proteins, we report hundreds of novel ones. Intriguingly, ∼40% of the secreted proteins were regulated under insulin-resistant conditions, including a protein family with signal peptide and EGF-like domain structure that had not yet been associated with insulin resistance. Finally, we report that secretion of IGF and IGF-binding proteins was down-regulated under insulin-resistant conditions. Our study demonstrates an efficient combined experimental and bioinformatics workflow to identify putative secreted proteins from insulin-resistant skeletal muscle cells, which could easily be adapted to other cellular models.

The present study investigated the effect of pomegranate extract (POMx) and pomegranate juice (POM juice) on the growth of major groups of intestinal bacteria: Enterobacteriaceae, Bacteroides fragilis group, clostridia, bifidobacteria, and lactobacilli, and the utilization of pomegranate polyphenols by Bifidobacterium and Lactobacillus. The total phenolic content of the pomegranate extract and juice was determined using the Folin-Ciocalteau colorimetric method and reported as gallic acid equivalent (GAE). The polyphenol composition was determined by HPLC. Stool specimens were incubated with 400, 100, and 25 μg/ml GAE POMx and POM juice and subjected to selective culture. Bifidobacterium and Lactobacillus strains were incubated with 400 μg/ml GAE POMx and POM juice and metabolites were analyzed. POMx and POM juice increased the mean counts of Bifidobacterium and Lactobacillus and significantly inhibited the growth of B. fragilis group, clostridia, and Enterobacteriaceae in a dose-response manner. Bifidobacterium and Lactobacillus utilized ellagic acid and glycosyl ellagic acid but little or no punicalin was utilized. Neither POMx nor POM juice was converted to urolithins by the test bacteria or the in vitro stool cultures. The effect of pomegranate on the gut bacteria considered to be beneficial (Bifidobacterium and Lactobacillus) suggests that pomegranate may potentially work as a prebiotic. The concept that polyphenols such as those in pomegranate impact gut microbiota populations may establish a new role for polyphenols in human health., (Published by Elsevier Ltd.)

Obesity is characterized by an excessive accumulation of body fat, for which impaired adipogenesis is believed to play a crucial role. As a gatekeeper of early adipogenesis, protein delta homolog 1 (DLK1) has a pivotal role in deciding whether pre-adipocytes will differentiate, determining the balance between healthy and unhealthy fat tissue. Here, an expression system for the cysteine-rich soluble human DLK1 was established. DLK1 was overexpressed in Escherichia coli BL21(DE3)pLysRARE, purified by affinity chromatography and refolded by stepwise dialysis. Identity, purity, secondary structure and refolding efficiency were determined. Proteolytic digestion followed by mass spectrometry analysis proved correct disulfide bridge formation. The biological activity of DLK1 was examined by differentiation assays in murine pre-adipocyte-like 3T3-L1 cells. Thereby, recombinantly produced DLK1 was shown to inhibit adipogenesis in a concentration- and time-dependent manner. All in all, our approach gives access to large amounts of active DLK1 and can be transferred to related proteins., (Copyright © 2015 Elsevier Inc. All rights reserved.)

To evaluate the clinic safety of human Midkine as an articular protective agent, recombinant mouse Midkine (rmMK) was prepared in prokaryotic system for the pre-clinic long-term studies in mice. The open reading frame of mouse Midkine (mMK) was sub-cloned onto expression vector pET30a (+) and transformed into Escherichia coli BL21 (DE3) strain line. The rmMK protein, with a Met fused at N terminus of native mMK for expression initiating, proved to be expressed in inclusion bodies and turned out to be soluble post-denaturation and renaturation. The soluble rmMK was purified successfully with ion exchange and affinity chromatography and characterised good enough to meet the requirements for animal use. Eventually, 13.2-mg rmMK with high quality and bioactivity was obtained from 1 L LB culture, and the total recovery was 11.4%. The present work laid a good foundation for pilot- or large-scale production of rmMK in prokaryotic system.

Background and Aims: It is suggested that regulatory immune cells play a critical role in cancer cell growth by facilitating cancer cells to escape from the immune surveillance. The generation of the immune regulatory cells in cancer has not been fully understood yet. This study aims to investigate the role of the hepatoma-derived growth factor (HDGF) in the generation of regulatory T cells (Treg)., Methods: CCL-9.1 cells (A mouse hepatoma cell line), were cultured. The expression of HDGF in CCL-9.1 cells was assessed by quantitative RT-PCR and Western blotting. The generation of Foxp3(+) T cells was assessed by cell culture and flow cytometry. The immune suppressor function of the Foxp3(+) T cells on CD8(+) T cell activities was assessed by the carboxyfluorescein succinimidyl ester (CFSE)-dilution assay and enzyme-linked immunosorbent assay., Results: The results showed that exposure to PolyIC markedly increased the expression of HDGF in CCL-9.1 cells. Coculture of CCL-9.1 cells and CD4(+) CD25(-) T cells in the presence of PolyIC generated the Forkhead box protein (Foxp)3(+) T cells. The exposure to HDGF increased the expression of Foxp3 and decreased the expression of GATA3 in CD4(+) T cells. After activation, the Foxp3(+) T cells suppressed the CD8(+) T cell proliferation and the release of the cytotoxic cytokines., Conclusions: Liver cancer cell-derived HDGF can induce Foxp3(+) T cells; the latter has the immune suppressor functions on CD8(+) T cell activities., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Low concentrations of black tea and water extracts from medicinal plants Arctostaphylos uva-ursi, Vaccinium vitis-idaea, Tilia cordata, Betula pendula and Zea mays stimulated biofilm formation in Escherichia coli BW25113 up to three times. Similar effect was observed for tannic acid and low concentrations of quercetin. In contrast, the extract from Urtica dioica reduced biofilm production. Pretreatment with plant extracts variously modified antibiotic effects on specific biofilm formation (SBF). Extract from V. vitis-idaea increased SBF, while the extracts from Achillea millefolium, Laminaria japonica and U. dioica considerably decreased SBF in the presence of ciprofloxacin, streptomycin and cefotaxime. Stimulatory effect of the extracts and pure polyphenols on biofilm formation was probably related to their prooxidant properties. The rpoS deletion did not affect SBF significantly, but stimulation of biofilm formation by the compounds tested was accompanied by inhibition of rpoS expression, suggesting that a RpoS-independent signal transduction pathway was apparently used.

CS5931 is a novel polypeptide from Ciona savignyi with anticancer activities. Previous study in our laboratory has shown that CS5931 can induce cell death via mitochondrial apoptotic pathway. In the present study, we found that the polypeptide could inhibit angiogenesis both in vitro and in vivo. CS5931 inhibited the proliferation, migration and formation of capillary-like structures of HUVECs (Human Umbilical Vein Endothelial Cell) in a dose-dependent manner. Additionally, CS5931 repressed spontaneous angiogenesis of the zebrafish vessels. Further studies showed that CS5931 also blocked vascular endothelial growth factor (VEGF) production but without any effect on its mRNA expression. Moreover, CS5931 reduced the expression of matrix metalloproteinases (MMP-2 and MMP-9) both on protein and mRNA levels in HUVEC cells. We demonstrated that CS5931 possessed strong anti-angiogenic activity both in vitro and in vivo, possible via VEGF and MMPs. This study indicates that CS5931 has the potential to be developed as a novel therapeutic agent as an inhibitor of angiogenesis for the treatment of cancer.

Phenolic compounds of nutraceutical importance viz., catechins (C), (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG) were estimated in fresh green tea shoots of Camellia sinensis (L) O Kuntze cultivar. The total polyphenols and total catechins were in the range of 219.90 to 317.81 and 140.83 to 271.39 g/kg, respectively in monthly samples of tea. The values of C, EC, EGC, EGCG and ECG in tea powders as analyzed through high performance liquid chromatography (HPLC) were in the range of 1.560 to 3.661, 13.338 to 27.766, 26.515 to 39.597, 62.903 to 102.168 and 18.969 to 39.469 mg/g, respectively. Effect of tea extracts and standard flavanols against five pathogenic bacteria viz., Listeria monocytogenes (MTCC-839), Pseudomonas aeruginosa (MTCC-741), Bacillus cereus (MTCC-1272), Staphylococcus aureus (MTCC-96) and Escherichia coli (MTCC-443), and eleven indigenous potential bacterial probiotics belonging to genera Enterococcus, Bacillus and Lactobacillus spp. obtained from fermented foods of Western Himalayas, was investigated. EGCG, ECG and EGC exhibited antibacterial activity but, C and EC did not show this activity. Tea extracts having high concentrations of EGCG and ECG were more potent in antibacterial action against bacterial pathogens. Tea extracts and standard flavan-3-ols augmented viability of potential probiotics in an order of EGCG > EGC > ECG > EC > C. Tea extracts and standard flavanols had no antibacterial activity against Escherichia coli (MTCC-443) but, in combination with probiotic culture supernatants, this activity was seen. The Kangra tea thus, exerts antibacterial effect on bacterial pathogens through EGCG, ECG and EGC constituents while stimulatory effect on growth of indigenous potential probiotics.

Sea urchins are an excellent model system for investigating fertilization mechanisms and fundamental cell biological phenomenon such as release from quiescence, cell division, secretion, and basic signal transduction. The ease of gamete collection, fertilization, and culture is complemented by exquisite developmental synchronicity and the ability to carry out both large-scale biochemical studies and single-cell experiments. In particular, fertilization in echinoderms serves as a paradigm for a digital signaling event-a one-time only switch that launches the egg into the developmental pathway. Sperm-induced egg activation is dependent on the release of calcium from internal stores and subsequent effects on a myriad of cellular events such as exocytosis, cytoskeletal remodeling, and cell cycle reentry. Here we describe methods to investigate individual signaling proteins as well as global proteomic and phosphoproteomic changes involved in the initial steps of egg activation through the egg-to-embryo transition.

Using bioinformatics analysis, we previously identified salusin-β, an endogenous bioactive peptide with diverse physiological activities. Salusin-β is abundantly expressed in the neuroendocrine system and in systemic endocrine cells/macrophages. Salusin-β acutely regulates hemodynamics and chronically induces atherosclerosis, but its unique physicochemical characteristics to tightly adhere to all types of plastic and glassware have prevented elucidation of its precise pathophysiological role. To quantitate plasma total salusin-β concentrations, we produced rabbit and chicken polyclonal antibodies against the C- and N-terminal end sequences, circumvented its sticky nature, and successfully established a sandwich enzyme-linked immunosorbent assay (ELISA). Salusin-β was abundantly present in the plasma of healthy volunteers, ranging from 1.9 to 6.6 nmol/L. Reverse phase-high performance liquid chromatography analysis showed that a single immunoreactive salusin-β peak coincided with synthetic authentic salusin-β. Plasma salusin-β concentrations were unaffected by postural changes and by potent vasopressin release stimuli, such as hypertonic saline infusion or smoking. However, salusin-β concentrations showed significant circadian variation; concentrations were high during the daytime and reached the lowest concentrations in the early morning. Plasma salusin-β levels in subjects with diabetes mellitus, coronary artery disease, and cerebrovascular disease showed distinctly higher levels than healthy controls. Patients with panhypopituitarism combined with complete central diabetes insipidus also showed significantly higher plasma salusin-β levels. Therefore, the ELISA system developed in this study will be useful for evaluating circulating total salusin-β levels and for confirming the presence of authentic salusin-β in human plasma. The obtained results suggest a limited contribution of the neuroendocrine system to peripheral total salusin-β concentrations and a role for plasma total salusin-β concentrations as an indicator of systemic vascular diseases.

For vision-threatening retinitis pigmentosa and dry age-related macular degeneration, there are no United States Food and Drug Administration (FDA)-approved treatments. We identified, biosynthesized, purified, and characterized lens epithelium-derived growth factor fragment (LEDGF1-326) as a novel protein therapeutic. LEDGF1-326 was produced at about 20 mg/liter of culture when expressed in the Escherichia coli system, with about 95% purity and aggregate-free homogeneous population with a mean hydrodynamic diameter of 9 ± 1 nm. The free energy of unfolding of LEDGF1-326 was 3.3 ± 0.5 kcal mol(-1), and melting temperature was 44.8 ± 0.2 °C. LEDGF1-326 increased human retinal pigment epithelial cell viability from 48.3 ± 5.6 to 119.3 ± 21.1% in the presence of P23H mutant rhodopsin-mediated aggregation stress. LEDGF1-326 also increased retinal pigment epithelial cell FluoSphere uptake to 140 ± 10%. Eight weeks after single intravitreal injection in Royal College of Surgeons (RCS) rats, LEDGF1-326 increased the b-wave amplitude significantly from 9.4 ± 4.6 to 57.6 ± 8.8 μV for scotopic electroretinogram and from 10.9 ± 5.6 to 45.8 ± 15.2 μV for photopic electroretinogram. LEDGF1-326 significantly increased the retinal outer nuclear layer thickness from 6.34 ± 1.6 to 11.7 ± 0.7 μm. LEDGF1-326 is a potential new therapeutic agent for treating retinal degenerative diseases.

Setaria digitata is an animal filarial parasite infecting cattle and other ungulates causing mild to severe diseases, and infection of human by this nematode has also been reported. Therefore, this study was undertaken as an initial step towards the characterization of an uncharacterized gene of S. digitata, that encode for parasitic nematode-specific growth factor-like protein to which Wucheraria bancrofti and other filarial and parasitic nematodes have homologous counterparts (revealed by bioinformatic analyses), by means of protein expression and purification in Pichia pastoris. In Pichia expression system, S. digitata uncharacterized protein (SDUP) was expressed both as secretory and intracellular protein. SDS-PAGE and western blot analyses revealed expression of recombinant SDUP in P. pastoris, showing a band of 23.8 kDa, which is similar to theoretical molecular weight and also this protein was shown to be more concentrated into insoluble fraction than soluble fraction of cell lysates suggesting that the protein to be more hydrophobic than hydrophilic. Recombinant SDUP was successfully purified using Ni-NTA affinity chromatography purification technique and three dimensional structural studies of the purified protein are currently being progressed. This is the first report of the expression of S. digitata protein in P. pastoris.

Cnidarian cell signalling remains poorly understood. This study has expanded our knowledge of the cell signalling molecule host release factor (HRF) from the coral Plesiastrea versipora. We have now confirmed that HRF is present in coral host cells that lack intracellular algae. Previous studies showed that HRF stimulates the release of photosynthetic products (mainly glycerol) from Symbiodinium algae, thus providing the host with carbon; glycerol release was accompanied by reduced synthesis of algal triacylglycerols and starch. In this study, we have shown that supplying glycerol to algae incubated with HRF does not restore normal triacylglycerol and starch synthesis. Release of (14) C-labelled products from algae may continue after photosynthesis ceases, although at a much lower rate. When algae were placed in the dark for 4 h with HRF following 2 h of photosynthesis in seawater, (14)C-labelled products were released, but at ≤ 15% of the amount released during 2 h of photosynthesis with HRF. HRF did not stimulate the release of compounds derived from a nonphotosynthetic source. The response of Symbiodinium from P. versipora to HRF has been compared with the response of Symbiodinium algae from Tridacna maxima, Heliofungia actiniformis, Aiptasia pulchella and Pocillopora damicornis to both their own HRF and to P. versipora HRF. Algae from P. versipora showed the highest response to both P. versipora HRF and to the other hosts' HRF. Further purification of P. versipora HRF suggests that HRF is a peptide with an acidic pI. We propose that HRF will provide a useful tool for the study of carbon metabolism., (© 2013 The Authors Journal compilation © 2013 FEBS.)

Growth factors and cytokines are active players in controlling the different stages of wound healing and tissue regeneration. Recent trends in personalized regenerative medicine involve using patient's own platelet-rich plasma for stimulating wound healing and tissue regeneration. This technology provides a complex cocktail of growth factors and even a fibrin scaffold with multiple biologic effects. In the last few years, an increasing number of studies provide evidence of the potential of combining platelet-rich plasma with different biomaterials in order to improve their properties, including handling, administration, bioactivity, and level of osseointegration, among others. In this review, we discuss the use of platelet-rich plasma as an alternative, easy, cost-effective, and controllable strategy for the release of high concentrations of many endogenous growth factors. Additionally, we provide an overview of the current progress and future directions of research combining different types of biomaterials with platelet-rich plasma in tissue engineering and regenerative medicine.

Human leukocyte cell-derived chemotaxin 2 (LECT2) is a chemotactic factor for neutrophils and a 16-kDa secreted protein consisting of 133 amino acids with three intramolecular disulfide bonds. Here, we propose an efficient method for the preparation of human LECT2 using a high hydrostatic pressure (HHP, 200 MPa) refolding technique. When LECT2 was over-expressed in Escherichia coli cells, most of LECT2 molecules became insoluble inclusion bodies (IBs). HHP was applied to the refolding of LECT2 from insoluble IBs, which dramatically improved the yield of the active LECT2. CD and NMR measurements demonstrated that the refolded LECT2 had a tertiary structure indistinguishable from the solubly expressed LECT2. In addition, both the refolded and solubly expressed LECT2 showed the same level of chemotactic activity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Growth factors (GFs) are chemical messengers that regulate specific cellular activities such as cell proliferation and formation of the extracellular matrix. GFs may be derived from a variety of sources, including animals., Objective: Evaluate the safety and efficacy of a topical antiphotoaging product containing secretions of the snail Cryptomphalus aspersa (SCA) for the improvement of facial rhytides., Materials and Methods: This was a 2-center, double-blind, randomized, 14-week study in which 25 patients with moderate to severe facial photodamage were treated with an emulsion (with 8% SCA) and liquid serum (with 40% SCA) on one side of the face and placebo on the contralateral side for 12 weeks. Silicone skin impressions of periocular rhytides were performed at baseline and after 12 weeks of treatment. Patient and physician assessments were also performed at 8, 12, and 14 weeks., Results: Periocular rhytides on the active ingredient side showed significant improvement after 12 weeks (P=.03) and improved texture to a greater degree than placebo at 8 and 12 weeks, as well as 2 weeks after discontinuing the product (14 weeks)., Conclusion: Daily application of topical products containing SCA proved effective and well tolerated for improvement in coarse periocular rhytides and fine facial rhytides. Subjects noted a significant degree of improvement in fines lines at the 8-week time point on the SCA-treated side (P≤.05) but did not report a significant difference in the quality of their skin.

Progranulin is a secreted glycoprotein, and the GRN gene is mutated in some cases of frontotemporal dementia. Progranulin has also been implicated in cell growth, wound healing, inflammation, and cancer. We investigated the molecular nature of secreted progranulin and provide evidence that progranulin exists as a homodimer. Although recombinant progranulin has a molecular mass of ∼85 kDa by SDS-PAGE, it elutes in fractions corresponding to ∼170-180 kDa by gel-filtration chromatography. Additionally, recombinant progranulin can be intermolecularly cross-linked, yielding a complex corresponding to a dimer (∼180 kDa), and progranulins containing different epitope tags physically interact. In plasma, progranulin similarly forms complexes of ∼180-190 kDa. Although progranulin partially co-fractionated with high density lipoproteins (HDL) by gel-filtration chromatography, we found no evidence that progranulin in mouse or human plasma is a component of HDL either by ultracentrifugation or by lipid binding assays. We conclude that circulating progranulin exists as a dimer and is not likely a component of HDL.

Lactoferrin is an iron-binding glycoprotein found in the milk of most mammals for which various biological functions have been reported, such as antimicrobial activity and bifidogenic activity. In this study, we compared the bifidogenic activity of bovine lactoferrin (bLF) and pepsin hydrolysate of bLF (bLFH), isolated bifidogenic peptide from bLFH, and investigated the bifidogenic spectra of bLF, bLFH, and its active peptide against 42 bifidobacterial strains comprising nine species. Against Bifidobacterium breve ATCC 15700(T), minimal effective concentrations of bLF and bLFH were 300 and 10 μg/ml. Against Bifidobacterium longum subsp. infantis ATCC 15697(T), the minimal effective concentration of bLFH was 30 μg/ml, and bLF did not show bifidogenic activity within 300 μg/ml. As an active peptide, a heterodimer of A(1)-W(16) and L(43)-A(48) linked by a disulfide bond was isolated. Previously, this peptide was identified as having antibacterial activity. An amino acid mixture with the same composition as this peptide showed no bifidogenic activity. The strains of each species whose growth was highly promoted (>150%) by this peptide at 3.75 μM were as follows: B. breve (7 out of 7 strains [7/7]), B. longum subsp. infantis (5/5), Bifidobacterium bifidum (2/5), B. longum subsp. longum (1/3), Bifidobacterium adolescentis (3/6), Bifidobacterium catenulatum (1/4), Bifidobacterium pseudocatenulatum (0/4), Bifidobacterium dentium (0/5), and Bifidobacterium angulatum (0/3). Growth of none of the strains was highly promoted by bLF at 3.75 μM. We demonstrated that bLFH showed stronger bifidogenic activity than natural bLF, especially against infant-representative species, B. breve and B. longum subsp. infantis; furthermore, we isolated its active peptide. This is the first report about a bifidogenic peptide derived from bLF.

We have developed a cysteine anchoring method for the synthesis of DILP8 and its analogues. The first is to synthesis of DILP8A SS13-18, C14-MeOBzl, C24-Acm and activate it as DILP8A S13-18, C14-SSPyr C24-Acm. A next step is to synthesize the DILP8BC16-Acm. The desired peptide, DILP8 with Cys(Acm) at A-24 and B-16, was then dissolved in 75% HOAc by addition of Iodine in MeOH and 4M HCl in dioxane. The reaction mixture was monitored by HPLC and the excess iodine was reduced with ascorbic acid. Purification of the peptide was achieved by HPLC. Pure synthetic DILP8 showed a single peak on analytical HPLC with corrected molecular ion. By using the above methods, enough peptide and highly homogenous pure DLP8 were generated.

Nucleotide-binding and leucine rich repeat domain-containing proteins (NLRs) are central to the formation of many inflammasome complexes. Several inflammasome forming NLR proteins are known to be ATPases, but the nucleotide binding specificity of many remains to be characterized. The oligomerization of NLR proteins and assembly of inflammasomes require the ATP (or other nucleotide) binding activity of the NLR proteins. Quantitative and qualitative studies of the nucleotide binding properties of these proteins are useful tools in studying the regulation of inflammasome activity, and are outlined in this Chapter.

The aim of this pilot study was to present a novel technique for the management of the Schneiderian membrane during maxillary sinus lift surgery using plasma rich in growth factors (PRGF). Eight maxillary sinuses were augmented in 8 patients. Two small perforations of the Schneiderian membrane occurred during the lifting procedure, which were solved using the PRGF clot before grafting the site with PRGF and anorganic bovine bone. With the exception of 1 patient who experienced pain following an acute sinus infection after 3 days of uneventful healing, the patients' postoperative quality of life was generally good. The most common complication (50% of cases) was hematoma, which disappeared after 1 week. Despite the limitations of this study concerning the sample size and the study design, the use of PRGF may be helpful in reducing complications following sinus lift surgery. More well-designed studies, with larger sample size, are needed to validate this protocol.

Polyphenolic extracts (PE) of edible flower of Sesbania grandiflora were tested to evaluate its antimicrobial effect against some common pathogenic bacteria and growth promoting property against probiotic organism Lactobacillus acidophilus. The antimicrobial activity of S. grandiflora flower PE against selected pathogens was evaluated using both in vitro and in situ methods. In vitro studies suggested that PE has inhibitory effect against Staphylococcus aureus, Shigella flexneri 2a, Salmonella Typhi, Escherichia coli and Vibrio cholerae. The gram-positive organism S. aureus was the most sensitive organism to PE and minimum inhibitory concentration (MIC) was found to be 0.013 mg/mL where as the MIC of PE against V. cholerae was the highest (0.25 mg/mL). On the other hand PE showed growth promoting effect on the common probiotic bacterium L. acidophilus. The major finding was that S. grandiflora PE induced a significant biomass increase of L. acidophilus grown in liquid culture media. PE showed reduction of S. aureus growth in food (fish) during storage at 10°C. High performance liquid chromatography analysis showed that rutin, a major flavonoid of the PE diminished in the culture medium MRS broth with the growth of L. acidophilus., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Background: Sessile marine animals such as sponges, ascidians, and bryozoans are a rich source of bioactive natural products, many of which exhibit potent anticancer activity., Materials and Methods: We extracted and purified a polypeptide with potent antitumor activity from Ciona savignyi by acetone fractionation, ultrafiltration, ion exchange chromatography, gel chromatography, and high-performance liquid chromatography. An MTT assay was used to study the cytotoxicity of the isolated fraction and the purified polypeptide. Cell cycle and Western blot analysis were performed to study the mode of action of the purified polypeptide., Results: A novel polypeptide with potent antitumor activity was purified. The molecular weight of the polypeptide, designated CS5931, was 5931 Da, and use of the genome basic local alignment search tool (BLAST) revealed that the N-terminal sequence of CS5931 is identical to that of granulin A from C savignyi. CS5931 exhibited significant cytotoxicity for several cancer cell types and induced apoptotic death in HCT-8 cells in a dose- and time-dependent manner. Cell cycle analysis demonstrated that CS5931 caused cell cycle arrest at the G(2)/M phase, and a sub-G(1) peak appeared after treating the cells with CS5931 for 12 hours. The mitochondrial-mediated pathway was implicated in CS5931-induced apoptosis., Conclusion: Our observations clearly demonstrate the antiproliferative and proapoptotic activities of the polypeptide CS5931 from C savignyi and the mitochondrial-mediated pathway involved in the polypeptide-induced cell death., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The UL16-binding proteins (ULBPs) are a novel family of human MHC class I-related, cell surface proteins that function as ligands for NKG2D. In this study, the gene encoding human ULBP3 was cloned into prokaryotic expression vector pQE30, resulting in a recombinant plasmid pQE30-ULBP3. The pQE30-ULBP3 was transformed into Escherichia coli M15 and induced the expression of recombinant protein ULBP3 (rec-ULBP3). The purified rec-ULBP3 as an antigen was used to immunize BALB/c mice. Through cell fusion, sub-cloning, and screening approach, three hybridoma cell clones expressing monoclonal antibodies (MAb) were acquired. The results from Western blot analysis, flow cytometry, and enzyme-linked immunosorbent assay showed that the hybridoma clones B2-F1-F1 and B4-C5-D11, and not G2-A4-A12, reacted with rec-ULBP3 and nature ULBP3 expressed on the cell surface of the tumor cells. In conclusion, the new MAb described here provides a valuable tool for further investigating ULBP3 function and clinical application.

Objective: To identify plasma protein biomarkers of cervical high-grade squamous intraepithelial lesion (HSIL) of Uyghur women by proteomics approach., Methods: Plasma protein samples of Uyghur women with HSIL and chronic cervicitis were analyzed with 2D HPLC followed by detection of target proteins with Linear Trap Quadrupole Mass Spectrometer (LTQ MS/MS)., Results: We detected three upregulated and one downregulated protein peaks representing protein constituents distinguishing HSIL from controls by 2D HPLC, identified 31 target proteins by LTQ MS/MS. Further confirmed analysis with online software IPA® 8.7 and ELISA assay showed APOA1 and mTOR as potential biomarkers., Conclusions: A distinct plasma proteomic profile may be associated with HSIL of Uyghur women.

Platelet-rich plasma (PRP) is a promising alternative approach based on the efficacy of autologous growth factors to accelerate tissue healing, allowing a fast recovery after muscles, ligaments, tendon or cartilage lesions. This literature review begin focusing on the role of platelets growth factors in these tissue healing and on the available preparation methods for PRP. Moreover we consider the in vitro and in vivo study on PRP, some of the most important therapeutic applications and limitations. Although several preclinical studies show promising results, clinical studies still show controversial results. Further studies are required to define the efficacy and to specify the way of using PRP in the orthopaedic practice.