Background: Worldwide, Streptococcus pyogenes is the leading cause of bacterial pharyngitis. To reduce the use of antibiotics, antimicrobial phytochemical-containing remedies, which have long been in use in traditional medicine, may provide new approaches for management of streptococcal pharyngitis. The objective of this study was to assess the inhibitory activities of 25 natural phenolic compounds against three strains of S. pyogenes. Methods: After an initial screening, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the nine most effective phenolic compounds were determined. The effect of four compounds with the lowest MIC and MBC on streptococcal growth and biofilm formation was also studied. Results: 1,2-Naphthoquinone and 5-hydroxy-1,4-naphthoquinone elicited the greatest anti-S. pyogenes activities with MICs ranging from 0.39 to 6.25 µg mL−1 and MBCs of 100 µg mL−1. Both naphthoquinones inhibited the biofilm formation at concentrations ranging from 12.5 to 50 µg mL−1. Biofilm reduction and altered bacterial cell structures were visible in scanning electron microscopy images of naphthoquinone-treated cells. Conclusion: In conclusion, 1,2-naphthoquinone and 5-hydroxy-1,4-naphthoquinone inhibit S. pyogenes and should be further investigated as candidates for the management of streptococcal pharyngitis. Streptococcus pyogenes [S. pyogenes are responsible for an estimated 600 million cases of throat infections per year [S. pyogenes throat infections, with macrolides being used for patients with β-lactam hypersensitivity. However, while resistance to β-lactams has so far not emerged in S. pyogenes, resistances were found for macrolides and some quinolones (fluoroquinolone) [S. pyogenes forms biofilm, which has been associated with antibiotic treatment failures [Pharyngitis or bacterial infection of the throat is a common disease, which is often caused by pyogenes ,2. Worldper year . β-Lactainolone) ,4. Moreofailures . Thus, tIn addition to treatment with prescribed medications, throat lozenges containing chemical anesthetics and antiseptics are being used by patients in the relief of the discomfort associated with the infection. The antimicrobial quaternary ammonium compound, dequalinium chloride, is similarly used in the treatment of common infections of the mouth and throat and incorporated in candy-based lozenge formulations . The antS. pyogenes, including polyphenols  and terpenes [Streptococcus species, including S. pyogenes, by phytochemicals through the prevention of the bacterial adherence of the pharynx, inhibition of glycolytic enzyme and pH drop, reduction of biofilm, and alteration of cell surface hydrophobicity [Natural products derived from plants have been used in traditional medicine since the ancient times and are, now, being widely studied for incorporation into mainstream products. Plant-derived compounds of interest are mostly secondary metabolites, which possess antimicrobial properties against microbial pathogens and spoilers . Recentlterpenes ,10,11,12S. pyogenes activities, which could be incorporated in dehydrated honey throat lozenges and similar products.The objective of the present study was to identify, among 25 different commercially available plant-derived phenolic compounds, the ones with anti-S. pyogenes strains: S. pyogenes ATCC ® 19615 ™, S. pyogenes ATCC ® 49399 ™ and a clinical isolate obtained from a patient with pharyngitis . The strains were routinely cultured in Brain Heart Infusion (BHI)  media at 37 °C under aerobic conditions. The experiments used the following human O-glucoside), one hydroxybenzoates , three hydroxycinnamates , two naphthoquinones , one stilbene (resveratrol), one tannin (tannic acid), and two chemical compounds (used as positive controls), one quaternary ammonium compound , and one substituted phenol (4-hexylresorcinol) were evaluated for their antibacterial properties. The compounds were purchased from Sigma-Aldrich Company . Before each experiment, the stock solutions were freshly made to a concentration of 10 mg mL−1 in 100% DMSO.Twenty-five phenolic compounds belonging to 12 different classes: three simple phenols , three isoflavones , one chalcone (phloretin), one chalcone glycoside (phlorizin), two flavan-3-ols , two flavanones (naringenin and hesperidin), two flavones (flavone and naringin), two flavonols (myricetin and quercetin hydrate), one flavonol glucoside (quercetin-3-−1) was initially assessed on S. pyogenes ATCC 19615 and ATCC 49399, using the broth micro-dilution assay as described by Clinical and Laboratory Standards Institute [−1 stock solution was added to 98 µL of BHI broth in order to reach a final test compound concentration of 100 µg mL−1. Finally, for each strain, 100 µL of standardized inoculum was added, resulting in an initial bacterial concentration of 5 log10 (CFU mL−1). Bacterial inocula in BHI broth (2 µL water) and BHI broth +1% DMSO were also run as a control. The growth of the bacteria was assessed after incubation for 24 h at 37 °C by reading the A600 nm using a plate reader . Each test was done in triplicate. In order to determine the most active compounds, a decrease in the A600 nm of 0.1 or more in the presence of the tested compounds in comparison to the control (bacteria inocula in BHI +1% DMSO) was considered to be indicative of an inhibition of the growth.The inhibitory effect of the phenolic compounds  in comparison with the control. To determine MBC, 30 µL from each well, where no growth was detected—i.e., compound concentration ≥MIC—was spread on BHI agar plates and incubated for 24–48 h at 37 °C. The lowest compound concentration resulting in no growth on the agar plate represented the MBC. Each sample was tested in triplicate and each experiment was repeated three times.MIC broth micro-dilution assay was performed as described in the previous section, except 2-fold dilutions were used to yield final test compound concentrations ranging from 0.19 to 100 µg mLS. pyogenes ATCC 19615 was quantified after 0, 2, 4, 6, 8, 10, and 24 h at 37 °C. At each time point, an aliquot (100 μL) was pipetted, serially diluted, and plated on BHI agar followed by enumeration and calculation of the mean log10 CFU mL−1 of the duplicate samples. Each time-kill experiment was repeated in three biologically independent assays (n = 6).The effect of concentrations ranging from 1 to 16 times MIC of the four most active compounds on the growth of S. pyogenes to form biofilm in the presence of 1,2-naphthoquinone, 5-hydroxy-1-naphthoquinone, dequalinium chloride, or 4-hexylresorcinol (0.19–100 µg mL−1) was assessed by measuring the metabolic activity of sessile biofilm cells using a 3--2,5-diphenyltetrazolium bromide (MTT) dye assay. After an exposure period of three days at 37 °C, the planktonic bacterial cells were eliminated by carefully removing the BHI medium from the wells. Adapted MTT assay was performed as described in Vybrant® MTT Cell Proliferation Assay Kit Protocol . After incubation for 10 min at 37 °C, the A540 nm was measured and used to calculate the minimum biofilm inhibitory concentration (MBIC), defined as the first concentration where the decrease in A540 nm was significant (p < 0.05) in comparison with the control.The ability of S. pyogenes ATCC 19615 biofilms formed (72 h at 37 °C) in the presence of MBIC concentrations of 1,2 naphthoquinone, 5-hydroxy 1,4 naphthoquinone, dequalinium chloride, 4-hexylresorcinol were prepared in microplates. The S. pyogenes biofilms were also grown in BHI broth (water) and BHI broth +1% DMSO as controls. After incubation, a sample fixation method for SEM [ for SEM  adapted t-test , p < 0.05) between test compound concentrations and controls were determined using the Student t-test on the A600 nm or A540 nm data, respectively.During the determination of the MIC and MBIC, statistically significant differences  of 1,2-naphthoquinone delayed growth during the first 10 h but after 24 h the bacterial concentration reached around 8 log10 CFU mL−1  after 24 h.Administration of 1.56 µg mL reached B. A bact−1 permitted growth and a twice-higher concentration of 0.78 µg mL−1 displayed a bacteriostatic effect. After 24 h, the concentrations of 1.56 and 3.125 µg mL−1 induced more than 2.5 log10 CFU mL−1 and 6.25 µg mL−1 caused a >4 log10-reduction (below the detection limit). For the dequalinium chloride C, the sa−1 caused a growth delay and a small log10-reduction (under 1 log10 CFU mL−1) after 24 h. However, the concentration of 25 µg mL−1 induced a log10-reduction of more than 3 log10 CFU mL−1 after 24 h  [S. pyogenes naphthoquinones possess two carbonyl groups in an aromatic ring as part of their quinone structure. This hypothesis is supported by a recent review which associated the oxydo-reduction activity of the quinone structure in 2-hydroxy-1,4-naphthoquinone with the generation of reactive oxygen species (ROS) and damage of macromolecules such as DNA, proteins, and lipids [Eremophila serrula, which also exhibited antagonism against S. pyogenes strain ATCC 10389 with low MIC (7.8 µg mL−1) and MBC (15.6 µg mL−1). According to the literature, the MICs of phenolic compounds against pharyngitis pathogens range from 1.52 to 100 µg mL−1 ,10,11,12. (2014)  stated td lipids . Ndi et Staphylococci and Streptococci thereby avoiding induction of the toxic shock syndrome [Rhodomyrtus tomentosa—appears to be slightly more effective: it exerted a faster bactericidal activity on S. pyogenes (within 5 and 6 h) at lower concentrations (6.5–12.5 µg mL−1) [Clinically, most antibacterials are described as potentially being both bactericidal and bacteriostatic. This was also observed in this study as the tested compounds. Even if a bactericidal action is preferred in the context of treatment, achieving a bacteriostatic effect may advantageously inhibit exotoxin production in syndrome . In compµg mL−1) . HoweverS. pyogenes has not been explored. The inhibition of biofilm formation is an interesting way to prevent the formation of well-organized attached bacterial biofilms and, thus, pharyngitis. We observed that the naphthoquinones elicited significant anti-biofilm activity against S. pyogenes, with metabolic MBICs in the same range as 4-hexylresorcinol and this was confirmed by SEM, where S. pyogenes ATCC 19,615 cells appeared less aggregated, showed morphological changes, and reduced biofilm formation. These observations are in accordance with the potential of phenolic compounds to damage the membrane structure as well as anti-S. pyogenes properties of phytochemicals such as anti-adhesion, anti-biofilm, and quorum-sensing inhibition [To the best of our knowledge, the anti-biofilm potential of naphthoquinones against hibition ,8. The gS. pyogenes. If the combination demonstrates a synergic and/or a complementary effect , it could become a combined treatment for patient’s pain relief. Moreover, it is also possible to incorporate the efficacious natural compounds in dehydrated honey lozenges. Honey itself has a recognized effect as being systemic antitussive and antimicrobial natural source [Considering the potential clinical application of our study, additional experiments could be conducted on combination of natural antibacterial agents and currently used antibiotics to enhance the present management practices against l source ,19,20. I


Undoubtedly, mast cells take part in host defense against microorganisms as they are numerous at the portal of infection, they release many proinflammatory and antimicrobial mediators, and they express pattern recognition receptors, such as TLRs. These receptors play a key role in recognition and binding molecules associated with microorganisms and molecules associated with damage. Cathelicidins exhibit direct antimicrobial activities against a broad spectrum of microbes by perturbing their cell membranes. Accumulating evidence suggests a role for these molecules in supporting cell activation. We examined the impact of human cathelicidin LL-37 on tissue mast cell TLR expression and distribution. Depending on context, we show that LL-37 stimulation resulted in minor to major effects on TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9 expression. Confocal microscopy analysis showed that, upon stimulation, TLRs may translocate from the cell interior to the surface and conversely. FPR2 and EGFR inhibitors reduced the increase in expression of selected receptors. We also established that LL-37 acts as a powerful inducer of CCL3 and ROS generation. These results showed that in response to LL-37, mast cells enhance the capability to detect invading pathogens by modulation of TLR expression in what may be involved FPR2 or EGFR molecules. Other potential LL-37 receptors are purinergic receptor P2X7, epidermal growth factor receptor (EGFR), or C-X-C chemokine receptor type 2 (CXCR2) [Cathelicidins, the family of highly diverse antimicrobial peptides, are found in many mammalian species including rabbits, horses, pigs, rats, monkeys, cattle, and humans. These natural antibiotics are composed of 12–50 amino acid residues, and their molecular weight are in the range of 3 to 10 kDa. Peptides from the cathelicidin family have phocytes . Moreovephocytes . Apart fphocytes . LL-37 cphocytes –10. Humaphocytes , 12. Furphocytes , 14. Inhphocytes . Additiophocytes . AlthougMast cells play an important role in host defense against a variety of pathogens. An important prerequisite for the role of mast cells in defense mechanisms is their location. Mast cells are especially numerous in the skin and they are present under the epithelium of the respiratory tract, gastrointestinal tract, and urogenital tract. In the tissues, these cells are very numerous near the blood and lymph vessels. Such a strategic location, practically in the gate of infection, allows them to very easily and quickly have contact with the pathogens penetrating the body , 18. FurMast cells express receptors which play a crucial role in recognition and binding both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) –27. ThesConsidering the important role of mast cells in the antimicrobial protection, it is essential to comprehend the regulation of TLR expression by these cells. Accordingly, we decided to evaluate the influence of cathelicidin LL-37, a key humoral factor of antimicrobial defense, on both surface and intracellular TLR expression in fully mature tissue mast cells.Female albino Wistar rats weighing 200–250 g, aged 3-4 months, were used. All animal experimental procedures were carried out in strict accordance with the recommendations in the act on the protection of animals used for scientific or educational purposes. The experimental procedures were approved by the Local Ethics Committee for Experiments on Animals of the Medical University of Lodz .6 cells/mL. Mast cells were prepared with purity > 98%, as determined by metachromatic staining with toluidine blue (Sigma-Aldrich). The viability of mast cells was over 98%, as estimated by trypan blue (Sigma-Aldrich) exclusion assay.Mast cells were collected from peritoneal cavities of rats by density gradient separation technique, as previously described , 29. Aftμg/mL in a humidified atmosphere with 5% CO2 at 37°C for 1, 3, or 6 h. For control, mast cells were incubated under the same conditions without LL-37. qRT-PCR was conducted as previously described [qRT-PCR was used to determine TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9 mRNA levels. Purified mast cells suspended in cDMEM were stimulated with LL-37  at a final concentration of 1 escribed , 28. Theμg/mL, for 1, 3, or 6 h in a humidified atmosphere with 5% CO2 at 37°C. After that, mast cells were fixed with CellFIX solution  for 15 min and washed twice with 1x PBS. Next, mast cells were resuspended in 1x PBS and stained for 1 h with appropriate antibodies (Santa Cruz Biotechnology Inc.). For control, mast cells were stained with isotype antibodies . Primary antibody was not added to the sample to certify nonspecific binding of the secondary antibody. Specificities of antibodies were confirmed using blocking peptides. Cells were then washed with 1x PBS and incubated for 1 h with Alexa 488-conjugated secondary antibodies  in 1x PBS. After that, mast cells were washed twice with 1x PBS and resuspended in 1x PBS. Stained mast cell fluorescence was measured with FACS Canto II flow cytometer (BD Biosciences).For determination of constitutive and LL-37-induced cell surface of TLR2, TLR4, and TLR5 and intracellular localization of TLR3, TLR7, and TLR9, flow cytometry technique was used. Constitutive expression of TLRs was assessed in freshly isolated native mast cells (nonstimulated cells). To determine the intracellular localization of TLRs, mast cells were previously permeabilized with 0.1% saponin (Sigma-Aldrich) for 30 min at room temperature. LL-37-induced receptor expression was estimated on mast cells incubated with LL-37, at a final concentration of 1 μg/mL, for 1, 3, or 6 h in a humidified atmosphere with 5% CO2 at 37°C. After that, mast cells were fixed with CellFIX solution for 15 min and washed twice with 1x PBS. Nonpermeabilized and permeabilized mast cells were stained for 1 h with appropriate antibodies (antibody dilution 1 : 100). Next, cells were washed with 1x PBS and incubated for 1 h with Alexa 488-conjugated secondary antibodies in 1x PBS. Finally, cells were washed and resuspended in 1x PBS. To confirm the specificity of primary antibody, isotype control was used. Primary antibody was not added to the sample to certify nonspecific binding of the secondary antibody.For determination of constitutive and LL-37-induced surface and intracellular distribution of TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9, confocal microscopy was performed. To determine the subcellular localization of TLRs, mast cells were previously permeabilized with 0.1% saponin for 30 min at room temperature. Constitutive expression of TLRs was assessed in nonstimulated cells. LL-37-induced receptor expression was estimated in mast cells incubated with LL-37, at a final concentration of 1 7R antagonist KN-62 , FPR2 antagonist PBP10 (Tocris Bioscience), and EGFR inhibitor AG1478  were used. Purified mast cells were pretreated with inhibitors or medium alone for 15 min at 37°C in a water bath with constant stirring, before the main procedure execution. KN-62 and PBP10 were used at concentrations of 1 μM, and AG1478 was used at a concentration of 5 μM. After that, cells were washed and incubated with LL-37 at 1 μg/mL for 3 h. The concentrations of all applied inhibitors were chosen in the preliminary experiments, in accordance with the manufacturer's instructions, and neither of the inhibitors affected mast cell viability, as examined by trypan blue exclusion assay. For control, mast cells were incubated with DMSO (Sigma-Aldrich) under the same conditions (vehicle-treated control). The same staining protocol was used as above.To determine the contribution of the LL-37 potential receptor, P2Xμg/mL or medium alone for 30 or 60 min in a humidified atmosphere with 5% CO2 at 37°C. Indicator for ROS, H2DCFDA (Thermo Fisher Scientific), was used at a concentration of 2 μM for 10 min. After that, cells were washed and resuspended in 1x PBS.To indicate ROS generation, mast cells were incubated with LL-37 at 1 Confocal microscopy method was conducted according to a previously described procedure . All sig2, 1 mM MgCl2, 10 mM HEPES buffer, 5.6 mM glucose, and 1 mg/mL BSA) were incubated with LL-37 at final concentrations of 1, 10, 20, or 40 μg/mL, mouse anti-rat IgE  at a final concentration of 5 μg/mL (positive control) or buffer alone (nonstimulated cells) in a water bath for 120 min at 37°C with constant stirring. The supernatants were collected by centrifugation. The concentration of CCL3 in supernatants was evaluated by ELISA kit , according to the manufacturer's instructions. The sensitivity of the assay was < 59 pg/mL.Purified mast cells suspended in the medium for rat mast cells  on each graph.The statistical analysis of experimental data was performed using Statistica 13 software . Data are presented as the mean ± SD. Normality of distribution was tested with the Shapiro-Wilk test. All comparisons between groups were carried out by using Student's μg/mL LL-37 by qRT-PCR. We found that mature mast cells constitutively express mRNA for all studied TLRs (P = 0.039) and TLR7 (P = 0.025).We first examined the expression of TLR mRNAs by mature rat mast cells in response to LL-37. TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9 transcripts were analyzed after 1, 3, and 6 h of stimulation with 1 ied TLRs , and theied TLRs . There wμg/mL or medium alone for 1, 3, or 6 h. As shown in the middle panel of P = 0.026) and TLR9 (P = 0.029).We were next interested in determining whether LL-37 stimulation influences TL receptor protein expression. Mast cells were incubated with LL-37 at a final concentration of 1 P < 0.001. After 6 h of incubation, the intensity of the signals from nucleus envelope was weaker but also detectable (161.8 ± 20.9 AU); P < 0.001.To assess location and distribution of TLRs, confocal microscopy technique was used. Mast cells were stained for surface and intracellular expression of all studied receptors. Isotype control and control for nonspecific binding of the secondary antibody confirmed the specificity of antibodies (data not shown). The changes in TLR2 expression are shown in P < 0.001. In turn, incubation with LL-37 for an extended time caused a decrease in TLR3 intracellular expression (125.2 ± 11.6 AU) in about 18% compared with nonstimulated cells (151.1 ± 10.9 AU); P < 0.001. Mast cell stimulation with cathelicidin resulted in an increase of signals both in the cell membrane and intracellular regions after 1 h which was documented by intensity diagrams beside each microphotograph. In turn, incubation with LL-37 for an extended time caused a decrease in TLR3 intracellular expression in about 50% compared with nonstimulated cells. Above observations are in good agreement with flow cytometric analysis. As demonstrated in P < 0.001. The intensity of the signals was the highest after 3 h of stimulation. The expression of TLR5 by mast cells is shown in P < 0.001.The expression of TLR3 by mast cells is shown in P < 0.001. The changes in TLR9 expression level are shown in P < 0.001. The same tendency was noticed for receptors localized in nucleus envelope at 3 h and the mean intensity amounted (231.1 ± 33.5 AU) in comparison to nonstimulated cells (97.9 ± 27.4 AU); P < 0.001.Single confocal sections, supplemented with fluorescence intensity diagrams, showed that in freshly isolated mast cells, TLR7 is localized mainly in the cell interior . LL-37 iμg/mL for 2 h, using medium alone or anti-IgE as negative and positive controls, respectively. After incubation, the levels of CCL3 in supernatants were determined by the specific ELISA kit. The results of these experiments are shown in μg/mL was the highest and up to 292.3 ± 22.48 pg/mL. We established statistically significant differences determined by one-way ANOVA (P = 0.025). LL-37-induced ROS generation by mast cells was evaluated by confocal microscopy. Mast cells were incubated with cathelicidin at the concentration of 1 μg/mL for 30 or 60 min. As shown in Next, we sought to determine whether LL-37 stimulation induces the mast cell proinflammatory response. To this end, mast cells were stimulated with LL-37 at the concentrations of 1, 10, 20, and 40 7R, FPR2, and EGFR in the activation of mast cells by LL-37. Therefore, we performed inhibitory experiments in which P2X7R antagonist KN-62, FPR2 antagonist PBP10, and EGFR inhibitor AG1478 were used. The signals obtained from confocal microscopy were validated with profile view image analyses (besides each microphotograph). We documented that mast cell pretreatment with the FPR2 antagonist and EGFR inhibitor noticeably and significantly suppressed LL-37-mediated TLR2 and TLR4 upregulation (Our goal here was to elucidate the involvement of surface molecules P2Xgulation . Mast ceThe course of the immune response to infection depends, among others, on many humoral factors, that is, complement system proteins, acute phase proteins, and alarmins including heat shock proteins, cytokines, interferons, and chemokines. The above-mentioned agents synthesized by many cells, in direct and indirect ways, modulate defense processes directed against pathogens. A significant group of humoral factors involved in defense mechanisms is the antimicrobial peptides, including primarily cathelicidins and defensins. Despite the multidirectional activities of cathelicidins, accumulating evidence suggests a role for these molecules in supporting cell activation.in vitro, including BMMCs, CBMCs, FSMCs, HCMCs, and PBMCs [in vivo rat mast cells isolated from the peritoneal cavity, that is, connective tissue-type mast cells. We established that these cells constitutively express mRNAs and proteins of all studied receptors. We clearly stated that TLR4 is located on the cell surface and that TLR7 and TLR9 are present mainly in the cell interior. It is worth pointing out that in resting mast cells, TLR2 and TLR5 were detected not only in the cell surface but also in the perinuclear region and that, although the TLR3 protein was predominantly found in the intracellular compartment, it is expressed in the cell surface, as well. Considering the important role of mast cells in host antimicrobial defense, these observations, particularly regarding TLR2, TLR3, and TLR5, are intriguing as compartmentalization of different TLRs imposes not only which ligands are recognized but also the type of cell response.It is well established that mast cells take part in defense mechanisms. Therefore, it is important to understand the influence of humoral factors involved in regulation of immune response to pathogen invasion on mast cell phenotype and activity. That is why we decided to assess the impact of cathelicidin LL-37 on the expression of TLRs, the molecules that play a pivotal role in innate immunity, in mast cells. Until today, TLR expression, at transcript and protein levels, was documented in different mast cell lines, such as HMC-1, KU812, LAD, and MC/9, as well in various mast cell differentiated nd PBMCs , 31. In Furthermore, we established that TLR expression and distribution in mast cells are strongly modulated by cathelicidin LL-37. Confocal imaging indicated that this peptide significantly upregulates TLR2 and TLR4 expression at the cell surface probably by inducing translocation of intracellular molecules to the cell membrane. Treatment with LL-37 caused the increase of TLR9 expression both at the cell surface and cell interior, as well. Microscopy analysis showed that LL-37 can cause translocation of TLR3 from the plasma membrane to the cytoplasm, as this peptide induced a transient increase of TLR3 in cell interior while surface expression of this receptor was decreased. Moreover, we documented that LL-37 treatment enhanced the intracellular expression of TLR5 and TLR7, presumably by increased synthesis of new molecules.β, IFN-γ, and TNF increases TLR2 mRNA expression [γ and M-CSF upregulate surface expression both TLR2 and TLR4 proteins on human peripheral blood monocytes [γ upregulates mRNA and both surface and intracellular protein levels of TLR4 in human mast cells. Yang et al. [Considering the significance of TLRs in the development of innate immunity processes, it seems to be of great importance to comprehend factors that influence TLR expression; however, there is little information on this subject. It was shown that treatment of murine macrophage cell line RAW264.7 with IL-2, IL-15, IL-1pression . Kurt-Jopression  stated tonocytes . It was onocytes  stated tg et al.  establisg et al. . It was g et al. . Only Yog et al.  evaluateβ-hexosaminidase release assessment. LL-37 activates LAD2 cells to LTC4 and PGE2 production and release [β, IL-2, IL-4, IL-5, IL-6, IL-31, TNF, GM-CSF, and chemokine CCL4 [β, CCL2, and CCL3, as well [There are data that LL-37 can directly activate mast cells to proinflammatory activity. It was documented that this peptide causes degranulation of both immature , 41 and  release  and stimine CCL4 , 41, 44. as well . Likewisμg/mL [μg/mL, respectively [in vitro, we used LL-37 at 1 μg/mL to assess antimicrobial peptide impact on TLR expression and concentrations from 1 to 40 μg/mL to determine whether LL-37 stimulation induces the mast cell CCL3 generation. Therefore, we can assume that concentrations of LL-37 applied in our studies reflect the cathelicidin physiological levels.To date, it has not been entirely clear what are the exact cathelicidin concentrations in physiological and pathological conditions. In healthy individuals, the LL-37 levels were as low as 39.9 pg/mL in sputum , 1–4 ng/μg/mL  and 4.8 ectively . In our + T lymphocytes, and eosinophils [7 purinergic receptor can be involved in LL-37-induced cell activation. This peptide, via P2X7 molecule, stimulates monocytes for IL-1β synthesis [4 and TXA2 synthesis [7, as Yoshioka et al. [7, might be the functional receptors for LL-37 on rat tissue mast cells.Up to date, it is not clear which surface molecule acts as a functional receptor for cathelicidin LL-37. There are data that LL-37 may act through FPR2, as this molecule is involved in the LL-37-mediated migration of neutrophils, monocytes, CD4inophils , 9; LL-3inophils ; and proinophils . Sun et inophils  documentinophils  documentinophils  noticed inophils . Few datynthesis  and actiynthesis  and macrynthesis . Zhang eynthesis  providedynthesis , 64. Subynthesis  and Yu eynthesis  indicatea et al.  indicateIn conclusion, we have provided evidence that LL-37 directly influences TLR level and distribution in mature tissue-resident mast cells. This peptide causes enhancement of TLR2, TLR4, and TLR9 on the mast cell surface and TLR3, TLR5, and TLR7 in the cell interior. TLRs play a crucial role in the early innate immune response as they recognize highly conserved microbial structural motifs as well as different endogenous molecules synthesized in response to microbial infection. Hence, LL-37 might enhance mast cell capability to detect invading pathogens and host DAMPs by modulation of TLR expression. These findings seem to be vital in view of mast cells' importance in host defense processes developed in response to infection.


Incompatible coefficient of thermal expansion (CTE) is supposed to be a reason for chipping of ceramic veneered zirconia. This study evaluates the effect of veneering ceramic at varied CTE on bond strength to zirconia.Zirconia disks  were prepared from Y-TZP (Cercon®) and sintered at 1350°C for 6 hours. All zirconia disks were veneered with ceramics  with varied CTE including VITADur® alpha (VDα), VITAVM®7 (VM7), VITAVM®9 (VM9), Cercon® ceramkiss (CCK), IPSe.max® ceram (IeC), and IPS dSIGN® (IdS) (n=15). The specimens were thermo-cycled  prior to determine the shear bond strength on a universal testing machine. The veneering ceramic and zirconia rods  were prepared for CTE evaluation. ANOVA and Tukey’s multiple comparisons were used to determine the statistically significant difference (α=0.05). Weibull analysis was applied for survival probability, Weibull modulus (m), and characteristics strength (σo) of the shear bond. The interfaces were microscopically examined. The phase transformation of zirconia was determined using X ray diffraction.p<0.05). The t→m phase transformation related with the CTE difference. The mean±sd (MPa), m, and σo of bond strength were 20.45±2.32, 9.25, and 21.53 for Z-VDα, 19.47±4.53, 4.66, and 20.31 for Z-VM7, 21.05±3.96, 5.61, and 21.88 for Z-IeC, 25.85±2.74, 9.93, and 27.15 for Z-VM9, 25.82±4.39, 6.27, and 27.06 for Z-CCK, and 2.96±0.73, 4.11, and 3.28 for Z-IdS. The CTE (×10-6/°C) were 10.80, 7.83, 7.87, 9.86, 9.93, 10.03, and 12.95 for Z, VDα, VM7, IeC, VM9, CCK, and IdS. The bond strength was significantly affected by the CTE difference (The CTE’s differences induced stress that affected the bond strength. CTE’s compatibility of veneering ceramic to zirconia is crucial for enhancing the bond strength. The CTE difference approximately 0.77-0.87×10-6/°C was recommended. Key words:Bond strength, coefficient of thermal expansion, zirconia. The celebrity of using all ceramic restoration has been increasing as they provide highly desirable esthetic appearance and extreme biological compatibility. Patients often request for metal-free restoration, which makes ceramic the most preferred restoration choice for reconstruction. Several new dental ceramics have been developed with improved strengths to withstand the stress from the physiologic masticatory function, which are being used as long-span fixed prostheses . Among cThe bond firmness between zirconia and ceramic that is partly related with the compressive force generated is because of appropriate difference of CTE’s value of veneering ceramic and zirconia -26. The The shear bond strengths of six ceramics, based on different CTE, including VITADur® alpha , VITAVM®7 , VITAVM®9 , Cercon®ceramkiss , IPSe.max® ceram , and IPS dSIGN®  veneered to Cercon® zirconia  were evaluated.-Zirconia Specimen PreparationNinety zirconia disks (12.0 mm in diameter and 1.2 mm in thickness) were prepared into disk shape from partially sintered Y-TZP blank using precision machine , and sintered in the furnace  at the recommended temperature of 1350ºC for 6 hours, to derive fully sintered zirconia disks (10.0 mm in diameter and 1.0 mm in thickness) due to 20% volumetric sintering shrinkage.-Ceramic Veneering TechniqueAll zirconia disks were randomly divided into six groups (15 disks per group) to be veneered with VDα, VM7, VM9, CCK, IeC, and IdS ceramic . The zir-Thermal Cycle TechniqueThe specimens were subjected to thermo-cycle process for 500 cycles between 5°C and 55°C for 30 seconds of immersion in each temperature bath, and 5 seconds for specimen transfer prior to the assessment of the shear bond strength.-Evaluation for Shear Bond StrengthEach specimen was evaluated for its shear bond strength by subjecting it to a compression shear apparatus on a universal testing machine . The shear load was applied at the zirconia-ceramic interface at crosshead speed of 0.5 mm/min. The loads at failure were recorded and calculated for shear bond strength  by dividing the failure load  by the area of interface , as shown in Equation 1: σ=P/A ……………….Equation 1.-Determination of Coefficient of Thermal ExpansionThe veneering ceramics and zirconia were prepared into a round rod shaped specimens . The creamy mixed consistency of veneering ceramic was placed, condensed into a silicone mold, and then removed for firing and glazing process. The zirconia rod was prepared from partially sintered Y-TZP blank and sintered in the furnace to derive the final dimension. The horizontal single rod dilatometer  was used to determine the heating and cooling curve for each specimen from 25°C to 500°C at the rate of 5°C per minute. The linear CTE was determined by using Proteus® software version 4.7 .Microscopic ExaminationThe de-bonding surfaces were examined under optical stereomicroscope  and scanning electron microscope  for characterizing the bonding failure’s mode. The zirconia-ceramic interface for each group was examined with SEM, and energy dispersive x-ray spectroscopy .-Evaluation of Crystalline StructureThe crystalline phases of ceramic veneered zirconia was determined for the relative amount of monoclinic (m) and tetragonal (t) phase of Y-TZP using the X-ray diffraction . The specimens were scanned with copper k-alpha (Cu Kα) radiation from the 2θ degree of 20–40o with 0.02o step size at every 2 seconds’ interval. The phase was analyzed in comparison to the known standard database of the joint committee on powder diffraction standards (JCPDS), and calculated for corresponding d-values using Bragg, as shown in Equation 2 : λ = d2 Where: λ is the X-ray wavelength (0.15418 nm for CuKa), d is normal distance of planes with the Miller indices (hkl), and θ is the Bragg angle.The ratio of m- to t- phase was determined by the peaks’ intensities using X’Pert Plus software . The mass fraction of m-phase to the total zirconia phase content was calculated from Garvie-Nicholson formula, as shown in Equation 3, and further corrected for nonlinearity using Toraya formula, as shown in Equations 4 and 5 , Fig. 11.Where: Im and It: integral intensities of monoclinic and tetragonal phaseC: composition-dependent correction factor (C = 1.32)Xt and Xm: the Toraya-corrected mass fraction of tetragonal and monolithic zirconia-Statistical Analysis of DataThe data was statistically analyzed using SPSS/PC Version 20 software . An analysis of variance (ANOVA) was used to determine the significant differences of shear bond strength upon different ceramics. Post-hoc Tukey HSD multiple comparison was used to determine the difference between groups at 95% level of confidence. Weibull analysis was performed to determine the bond strength’s reliability using Weibull++®statistics , and estimated the Weibull modulus (m) from Equation 6 and from a slope of the straight line plotted between ln{ln(1/Ps(Vo)} against m ln(σ/σo). Ps (VΟ)=exp{-(σ⁄σΟ)m } …………..Equation 6Where: Ps (Vo) is the probability of survival as the fraction of identical sample; Vo is the volume of the sample;σ is the shear strength; σo is the Weibull characteristic strength; and m is Weibull modulus.p<0.05) (p<0.05), except for the groups of Z-VM7, Z-VDα, and Z-IeC (p>0.05)  . Post-ho(p>0.05) . Weibull(p>0.05) B,D,F,H,JThe x-ray diffraction patterns revealed that most of crystal structure of tetragonal (t) phase with minor amount of monoclinic (m) phase in every group as shown in Figure This study indicated that bond strength of ceramic veneered zirconia is affected by veneering ceramics that possess different CTE values. Thus, null hypothesis was rejected. The CTE displays a major role in bond strength of ceramic veneered zirconia ,18. The The CTE’s difference principally influenced the interfacial residual stress that reflected on the amount of t→m phase transformation of zirconia . The resTo generate suitable levels of residual stress for promptly bond strength, appropriate CTE difference need to be primarily considered . This stThis investigation described the role of CTE in bond strength of ceramic veneered to Y-TZP. The study proved that bond strength of ceramic veneered zirconia was influenced by the CTE’s difference between zirconia and veneering ceramic. An extreme CTE difference between veneering ceramic and zirconia did not render favorable bond, but resulted in a higher stress inducing phase transformation of zirconia to facilitate the ease of bond failure. Proper selection of veneering ceramic based on appropriated CTE difference is extremely crucial to assure durable bond strength of ceramic fused with zirconia. The CTE of veneering ceramic less than that of zirconia for approximately 0.77-0.87X/ oC was suggested to provide suitable residual compressive stress for conferring a favorable bond strength.


Especially, an excellent cycling stability are achieved, a high discharge capacity of 890 mAh g−1 retained at a rate of 1.0 A g−1, suggesting its promising applications in lithium ion batteries (LIBs).The 3D flowerlike iron sulfide (F-FeS) is successfully synthesized via a facile one-step sulfurization process, and the electrochemical properties as anode materials for lithium ion batteries (LIBs) are investigated. Compared with bulk iron sulfide, we find that the unique structural features, overall flowerlike structure, composed of several dozen nanopetals and numerous small size iron sulfide particles embedded within the fine nanopetals, and hierarchical pore structure features provide signification improvements in lithium storage performance, with a high-rate discharge capacity of 779.0 mAh g Amo−1 . Therefo+ and electronic transport path lengths (permitting operation with low Li+ and electronic conductivity or higher power) [Nanostructure materials are becoming increasingly important, and have attracted great attention, due to the unusual electrical and mechanical properties endowed by confining the dimensions of such material, and due to the combination of surface and bulk properties to the whole behavior ,9,10,11.r power) ,14,15,16r power) ,18,19.9S8/Co1−xS@NC, with a discharge capacity of 813 mAh g−1 delivered at a current density of 2.0 A g−1 [2@C composites, delivering a discharge capacity of 697 mAh g−1 at a current density of 5 A g−1 [2O4, delivering a discharge capacity of 420 mAh g−1 at a rate of 2 A g−1 [Recently, considerable interest has focused on materials with three-dimensional (3D) hierarchical nano/microstructures, due to the 3D architecture providing a highway for the diffusion of both electrolyte and electron, which can facilitate efficient energy storage. Among these, materials with 3D flowerlike structure have been widely used as anodes in LIBs. For example, Wang et al. prepared flowerlike Co.0 A g−1 . Hu et a 5 A g−1 . Li et a 2 A g−1 . Therefo−1 at a high rate of 5 A g−1) and cycling performance (890.0 mAh g−1 retained after 100 cycles at a rate of 1 A g−1), further highlighting its tremendous potential for commercial application.However, iron sulfide based materials with three-dimensional (3D) flowerlike hierarchical nano/microstructure were rarely reported. Besides, iron sulfide based material possessed a high energy/power density and abundant reserves in the earth, having also attracted considerable attention as alternative promising anode materials for LIBs ,24. Base3·6H2O, 250 mM (2.7 g) urea, and 124 mM (7.2 g) tetrabutylammonium bromide were dissolved into 180 mL ethylene glycol (EG) in a 250 mL round-bottom flask. The red solution was obtained after chemicals completely dissolved. Then, the solution was gradually heated to refluxing temperature (ca. 185 °C) under stirring. After 12 min, a yellow precipitate appeared, and then the mixture turned yellow-green immediately. After 8 min, the mixture turned completely green, indicating the formation of the 3D flowerlike hierarchical nanostructure. After refluxing for 30 min, the reaction was ceased, and the mixture was cooled at ambient condition. After cooling to room temperature, the green precipitate was collected, and rinsed thoroughly with ethanol three times, before thoroughly drying at 60 °C in air.All the chemicals were directly used without further purification. In a typical synthesis with some modifications, 25 mM (1.2 g) FeCl−1 under Ar atmosphere. Finally, the FeS with 3D flowerlike nanostructure were obtained. As comparison, one sample was annealed under air atmosphere at 450 °C for 5 h with a ramp of 3 °C min−1, before the sulfurization process. The sulfurization process was the same as mentioned earlier. After that, bulk FeS (B-FeS) was synthesized.Briefly, the obtained precursor and sulfur (with a weight ratio 1:3) were loaded into two separate alumina boats, then transferred to a tube furnace for sulfurization at 600 °C for 5 h, with a heating rate of 1 °C minα radiation, λ = 1.54178 Å). Field-emission scanning electron microscopy , and transmission electron microscope  were used to investigate the morphology and microstructure of the as-obtained F-FeS and B-FeS. Thermogravimetric (TG) analysis was measured on a TG apparatus  in the temperature range of 25–900 °C at a ramp of 10 °C min−1 in air. The Brunauer–Emmett–Teller (BET) method was utilized to investigate the specific area by N2 adsorption–desorption isotherms. To further characterize the chemical composition, XPS analysis was performed with X-ray photoelectron spectroscopy . Raman spectra was also performed to investigate the existence of carbon.The crystal structures were characterized by X-ray diffraction analysis (XRD Rigaku D-max-γA XRD with Cu KN-methyl-pyrrolidone (NMP) to produce electrode slurry. The slurry was uniformly pasted onto pure copper foil current collector. After drying at 120 °C in a vacuum oven for 6 h, the Cu foil coated active material was used as working electrode, with metallic lithium foil as the counter electrode and separated by a Celgard 2500 membrane separator. The cells were assembled in a glove box filled with pure argon. After shelving for 12 h, the galvanostatic discharge–charge measurements were conducted on LAND CT2001A battery tester at various current densities over a voltage range of 0.0–3.0 V (vs. Li+/Li). Cyclic voltammetry (CV) curves and electrochemical impedance spectroscopy (EIS) were measured on the Autolab PGSTATN302N electrochemical workstation . Cyclic voltammetry (CV) tests were carried out at a scanning rate of 0.1 mV s−1 from 0.0 to 3.0 V. For the electrochemical impedance spectroscopy (EIS), the measurements were performed over a frequency range from 100 KHz to 10 mHz with 10 mHz. This section may be divided by subheadings. It should provide a concise and precise description of the experimental results, their interpretation, as well as the experimental conclusions that can be drawn.Electrochemical performances were tested in CR2032-type coin cell at room temperature. For preparing electrode, the active material was mixed with carbon black (Super-P) and poly(vinyl difluoride) (PVDF) binder with a mass ratio of 70:20:10, then dissolved in 2O3 , as shown in The unique 3D F-FeS nanostructure has been successfully synthesized via a simple one-step sulfurization reaction at 600 °C under Ar atmosphere. Particularly, 3D flowerlike Fe-based precursors were prepared according to previous literature, with some modifications; X-ray diffraction pattern XRD;  showed t2O3 obtained after the annealing process in air. The flowerlike architecture is perfectly inherited, and the morphology of nanopetals has no significant structural breakage or collapse, as shown in The morphology and microstructure of the as-prepared products were investigated by field-emission scanning electron microscopy (FE-SEM) as illustrated in 2 physisorption measurements were carried out. As shown in 2 g−1, than that of the B-FeS (13.3 m2 g−1)  . The por3/2 and Fe 2p1/2, respectively, indicating the existence of Fe2+ in 3D F-FeS. Two satellite peaks were observed at ~719.25 and 732.78 eV. The signal centered at ~706.78 eV was maybe attributed to a little of metallic Fe. Additionally, peaks observed at ~711.27 and 726.74 eV were ascribed to Fe3+, due to the Fe2+ partly oxidizing in air on the surface of material [3/2 and S 2p1/2, respectively, the characteristic peaks of FeS, whereas another peak at ~168.41 eV probably corresponded to oxidized group (SOx), due to the sulfur species being partly oxidized in air on the surface of material [Besides, X-ray photoelectron spectroscopy (XPS) was performed to further investigate the information on the surface chemical compositions and valence states of the 3D F-FeS. material . The simx2−FeS2 (0 < x < 2) (Equation (2)). The shape of CV curves changes after the first cycle, suggesting that the redox reactions undergo changes in the subsequent cycles. In the subsequent lithiation cycles, the cathodic peaks appearing at 1.86 V was attributed to the step-by-step formation of Li2FeS2 from Lix2−FeS2 (0 < x < 2) phase. The anodic peaks appearing in the subsequent cycles were contributed to the delithiation process from Lix2−FeS2 (0 < x < 2) to Li2FeS2. The reaction process between Lix2−FeS2 (0 < x < 2) and Li2FeS2 is reversible [In order to evaluate the potential use of 3D F-FeS nanostructures as anode for LIBs, as-prepared materials were assembled into half-cells and evaluated by a series of electrochemical measurements. Cyclic voltammetry (CV) was applied to investigate the redox reactions taking place in 3D F-FeS and B-FeS. versible ,33,34. Fversible . The ano−1. The 3D F-FeS delivered an initial discharge capacity of 1262.9 mAh g−1, and a charge capacity of 991.2 mAh g−1 at a current density of 0.1 A g−1, giving an irreversible capacity loss of 271.7 mAh g−1 and a Coulombic efficiency of 78.5%. The initial capacity loss can be attributed to the inevitable formation of solid-electrolyte interface (SEI) layer and initial irreversible lithium consumption. At the current densities of 0.2, 0.5, 1.0, 2.0 A g−1, it delivered discharge capacities of 973.3, 949.3, 900.7, 854.9 mAh g−1. At a high current density of 5 A g−1, 3D F-FeS still delivered a discharge capacity of 779.0 mAh g−1, much higher than that of B-FeS  layer, respectively. In the subsequent discharge curves, the slope between 0.88 and 0.8 V disappeared, a small slope showed around 1.9 V. These phenomena are consistent with the CV analysis. Compared with B-FeS, the 3D F-FeS exhibited outstanding high-rate capability as an anode material in LIBs  and constant phase element (CPE) between electrode/electrolyte interface [ct of 3D F-FeS electrode is 97.89 Ω, which is much smaller than that of B-FeS (114.7 Ω). This fact, once again, validated that the unique flowerlike structure and small size FeS particles enhanced rapid electron transport during the process of lithiation/delithiation reactions, improving the electrochemical performances significantly. The EIS spectra after 100 cycles is shown in To further understand interfacial properties of the 3D F-FeS with respect to the B-FeS electrode, electrochemical impedance spectroscopy (EIS) measurements were taken. As shown in nterface ,39,40. Cnterface , and Tab−1, and the 3D F-FeS material still sustains the original flowerlike framework, exhibiting its structure stability during the lithiation and delithiation process. Meantime, compared with 3D F-FeS material before cycling  the small size iron sulfide particles in the nanopetals could shorten the diffusion path length of lithium ions and alleviate the volume strain during the lithiation/delithiation process, which effectively promote the high rate capability and cycling stability; (2) the fine nanopetals assembled by small size iron sulfide particles possess dual-channel structure, greatly promoting the fast transportation for both lithium ions and electrons, which make the iron sulfide active material fully utilized; (3) the 3D flowerlike structure facilitated the penetration of electrolyte into electrode, due to its hierarchical pore structure and high specific surface area.−1 at a current density of 5 A g−1. In particular, an excellent cycling stability can be achieved at a current density of 1.0 A g−1 up to 100 cycles, even retaining a high capacity of 890 mAh g−1, confirming the 3D flowerlike iron sulfide as a promising anode material for LIBs.In summary, the 3D flowerlike iron sulfide (F-FeS) with high phase purity were facilely synthesized through one-step sulfurization of Fe-based precursors, and used as anode material in LIBs. The overall flowerlike structural features composed of several dozen nanopetals were perfectly inherited after sulfurization process and numerous small size iron sulfide particles embedded within the fine nanopetals. Benefiting from the unique structural features and hierarchical pore structure, the 3D flowerlike iron sulfide (F-FeS) electrodes manifested impressive lithium storage properties with a high-rate discharge capacity of 779.0 mAh g


Metastasis is a leading cause of death among cancer patients. In the tumor microenvironment, altered levels of extracellular matrix proteins, such as collagens, can facilitate the first steps of cancer cell metastasis, including invasion into surrounding tissue and intravasation into the blood stream. However, the degree of misexpression of collagen genes in tumors remains understudied, even though this knowledge could greatly facilitate the development of cancer treatment options aimed at preventing metastasis.We systematically evaluate the expression of all 44 collagen genes in breast cancer and assess whether their misexpression provides clinical prognostic significance. We use immunohistochemistry on 150 ductal breast cancers and 361 cervical cancers and study DNA methylation in various epithelial cancers.COL4A1 and COL4A2 overexpression and COL17A1  underexpression provide independent prognostic strength . Immunohistochemistry on ductal breast cancers confirmed that the COL17A1 protein product, collagen XVII, is underexpressed. This strongly correlates with advanced stage, increased invasion, and postmenopausal status. In contrast, immunohistochemistry on cervical tumors showed that collagen XVII is overexpressed in cervical cancer and this is associated with increased local dissemination. Interestingly, consistent with the opposed direction of misexpression in these cancers, the COL17A1 promoter is hypermethylated in breast cancer and hypomethylated in cervical cancer. We also find that the COL17A1 promoter is hypomethylated in head and neck squamous cell carcinoma, lung squamous cell carcinoma, and lung adenocarcinoma, in all of which collagen XVII overexpression has previously been shown.In breast cancer, various tests show that COL17A1 promoter methylation status accurately predicts both the direction of misexpression and the increased invasive nature for five out of five epithelial cancers. This implies that aberrant epigenetic control is a key driver of COL17A1 gene misexpression and tumor cell invasion. These findings have significant clinical implications, suggesting that the COL17A1 promoter methylation status can be used to predict patient outcome. Moreover, epigenetic targeting of COL17A1 could represent a novel strategy to prevent metastasis in patients.Paradoxically, collagen XVII is underexpressed in breast cancer and overexpressed in cervical and other epithelial cancers. However, the The online version of this article (doi:10.1186/s13148-016-0290-6) contains supplementary material, which is available to authorized users. Metastasis, the spread of cancer cells to distant organs, is one of the leading causes of death among cancer patients. To be able to disseminate, cancer cells need to overcome a number of barriers. In epithelia, cell-cell interactions and a basement membrane initially constitute major obstacles. In addition, once local invasion through the basement membrane has occurred, tumor cells need to be able to survive in the very different environment of the stroma .The stroma consists of fibroblasts and extracellular matrix (ECM). The ECM is composed of polysaccharides, water, and stromal cell-secreted proteins, as well as soluble growth factors sequestered by matrix components . Two typWhile initially regarded as a physical barrier to tumor cell migration, recent studies have shown that collagens also support tumor progression depending on the stage of cancer development . AssociaIn contrast to other collagens, collagen types XIII, XVII, XXIII, and XXV are transmembrane proteins, characterized by an N-terminal cytoplasmic domain and an extracellular C-terminus that contains 3 to 15 collagenous domains . Most reCOL17A1, the gene that encodes collagen XVII, is most significantly associated with poor patient prognosis. Consistently, collagen XVII levels are reduced in breast tumors and this is strongly associated with tumor stage, invasion, and menopausal status. Conversely, collagen XVII levels are elevated in cervical cancer and this is associated with increased local metastasis. Interestingly, the COL17A1 promoter methylation status correctly predicts the direction of collagen XVII misexpression in multiple types of epithelial cancers, including breast and cervical cancer.Here, we systematically study the expression of all 44 collagen genes in breast cancer. We find that reduced expression of p < 0.05, 1052 < n < 4177; Additional file To identify collagens whose misexpression may contribute to breast cancer development, and in particular metastasis, we systematically evaluated expression levels of all 44 collagen genes. By combining microarray expression data from 26 previously published datasets, Cox proportional hazard analyses were performed based on expression level and distant metastasis-free survival see the “” sectionp < 0.05   , 18. ThiCOL4A1 and COL4A2 and underexpression of COL17A1 correlating with poor distant metastasis-free patient survival  and that COL4A2 expression was significantly reduced , rather than increased  [p < 0.0001, n = 1075; Fig. COL17A1.In an effort to independently validate these results, we used a combination of 28 other microarray datasets, as described , 20. Howsed Fig. . Only COs (TCGA) , 22 alsoCOL17A1 expression is associated with poor distant metastasis-free survival, recurrence-free survival, and overall patient survival, as previously described [p = 0.0044, n = 1601; p = 6.58 × 10−10, n = 3524; p = 0.0341, n = 1115, respectively, log-rank test; Fig. In addition to the above survival analyses  using a previously well-characterized antibody [p = 0.1908, Fisher’s exact test). Neither was the decrease in the fraction of moderately stained sections (from 9% (5/57) to 3% (4/150), p = 0.1185; Fig. Focusing on protein-level expression of collagen XVII, the product of the antibody , 16. We p = 0.0038, Fisher’s exact test; Table p = 0.2308).Consistent with our above prognostic analyses, the ductal breast carcinomas stained positive for collagen XVII significantly less frequently that normal control tissue (22% 33/150) vs 42% (24/57), 50 vs 42%p = 0.0273) was further reduced significantly to 10% (4/39) in late stage cancers . The fractions of tumors that stained positive also significantly declined as tumors become more invasive, from 28% (27/97) in tumors that only locally invaded the submucosa and/or muscle (T1/2) to 11% (6/53) in tumors that invaded through underlying muscle and/or into other organs (T3/4) (p = 0.0143). Only 8% (2/25) of the most invasive cancers stained positive (p = 0.0015 compared to 42% (24/57) in normal tissue). Collagen XVII positivity also reduced with an increase in the number of positive lymph nodes and metastasis. Yet, this trend was not statistically significant , progesterone receptor (PR), or HER2 amplification status Table . Howeverp = 0.0046, Mann-Whitney U test, n = 185; Fig. p = 0.0442, Fisher’s exact test), moderately to strongly positive (23% (7/31) and 44% (146/331), respectively; p = 0.0146), or strongly positive (0% (0/31) and 20% (65/331), respectively; p = 0.0016) , lost one or both alleles. This led to a significant reduction of COL17A1 mRNA levels compared to diploid tumors , whose levels were already significantly lower than in normal breast tissue .The misexpression of collagen XVII in breast and cervical cancers in opposite directions, both at mRNA and at protein levels, prompted us to investigate whether changes in allelic copy numbers could provide an explanation for this. We used SNP array GISTIC copy number data and RNAseq data from the respective TCGA studies , 22. WhiCOL17A1 expression levels in breast cancer suggests that additional mechanisms regulate this gene’s expression. Tumor cells may silence gene expression through promoter hypermethylation [COL17A1 promoter methylation status in normal breast and breast cancer samples using TCGA DNA methylation data [COL17A1 promoter is indeed hypermethylated in breast tumors  precluded accurate analysis. For the remaining cancers, we find that the degree of COL17A1 promoter methylation is significantly reduced in the tumor samples  compared to normal control samples .Interestingly, collagen XVII overexpression is observed by IHC in various other cancers, including skin SCC, melanoma, non-small cell lung cancer, lung adenocarcinoma, lung SCC, and head and neck SCC , 25–28. p < 0.0001, Mann-Whitney U test; n = 497 and n = 112, respectively). Also, in normal cervix, COL17A1 levels do not significantly differ from those in breast carcinoma , whereas the levels in normal cervix are significantly lower than in normal breast tissue  were performed using probe 204636_at and the median as the cut-off between low and high expression, as described [Validation analyses for escribed , 20. In escribed , 22 wereescribed . Log-ran2O2 for 10 min, washed 2 × 5 min with PBS, incubated with HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody  in blocking buffer for 1 h, followed by 2 × 10 min PBS washes. TMAs were then stained with 3,3′-diaminobenzidine (DAB) reagent  and counter-stained with hematoxylin. Stepwise dehydration occurred in an ethanol series , followed by 9 min baking at 60 °C and incubation in 100% xylene for 2 min. Slides were mounted using Permount and dried overnight. Slides were imaged using Olympus Slide scanner VS120 (rm4026) and OlyVia software. Tissue sections were excluded from the analyses if chronic mastitis (breast), chronic inflammation/cervicitis, or cataplasia (cervix) was diagnosed or if more than 70% of the section was missing. Slides were independently scored by two individuals and in a blinded fashion. Clinical endpoints examined included pathology, age, grade, stage, tumor invasion, lymph node status, metastasis and estrogen (ER), progesterone (PR), and HER2 receptor status. Fisher’s exact tests were used for statistical analyses. In addition, we controlled for multiple testing by subjecting our analyses to a false discovery rate (FDR) of 5%, as previously described [Paraffin-embedded breast cancer and cervical tissue microarrays (TMAs) were obtained from US Biomax Inc. . Samples were obtained under the Health Insurance Portability and Accountability Act (HIPAA)-approved protocols, in accordance with the approved guidelines and with informed consent from the donors. For the breast cancer analysis, only patients with ductal carcinoma pathology were included. However, no inclusion or exclusion criteria were applied based on treatments received. Cores were 5 μm thick and had a 1 mm diameter. TMAs were sectioned and stored at 4 °C until use. TMA slides were baked at 60 °C for 30 min, incubated in 100% xylene for 10 min for de-paraffinization, and incubated in ethanol series  and milliQ water for 10 min each for rehydration. For antigen retrieval using sodium citrate buffer , slides were placed in a water bath, microwaved for 5 min (at P100/high), cooled for 5 min at room temperature (RT), and microwaved for 5 min. Slides were then cooled under tap water for 6 min, washed 2 × 5 min with PBS, and permeabilized in PBS/0.01% Triton-X for 10 min. Following 2 × 10 min PBS washes and 1 h incubation in blocking buffer (PBS/10% FBS), slides were incubated in blocking buffer with primary anti-collagen XVII antibody   overnigh VS120 rm26 and OlCOL17A1 allelic copy numbers were determined using Affymetrix Genome-Wide SNP6.0 Array datasets and GISTIC 2.0. RNAseq data, obtained from the TCGA breast cancer, cervical cancer, head and neck SCC, lung adenocarcinoma, and lung SCC Illumina HiSeq RNA Seq V2 (RSEM) datasets [U tests were used to compare differences.Putative datasets , 41–43. COL17A1 promoter methylation analyses, Illumina Infinium HumanMethylation450 platform level 3 data were used from the respective TCGA cancer datasets [β values for all probes in the region from 400 bp upstream to 400 bp downstream of the COL17A1 transcription start site (chromosome 10/hg19 genomic coordinate 105845638) were used , Table S2 , Table S3 , Figure S1 , and Supplementary References. (PDF 1136 kb)Supplementary Material containing Additional file 2:COL17A1 promoter methylation levels . (XLSX 231 kb)


Per2, Bmal1, Rev-erbα, and Dbp) were measured after an ex vivo challenge with several PAMPs and to further test the relevance of PAMP alteration of the molecular clock, an in vivo poly(I:C) challenge was performed. This study revealed that several other PAMPs are also capable of altering clock gene expression.Circadian rhythms are endogenous 24-h oscillations that influence a multitude of physiological processes. The pathogen-associated molecular pattern (PAMP), lipopolysaccharide, has been shown to modify the circadian molecular clock. The aim of this study was to determine if other PAMPs alter clock gene expression. Therefore, mRNA levels of clock genes ( Period (Per1-3), Cryptochrome (Cry1-2), Bmal1 (brain and muscle ARNT-like 1) and Clock [Rev-erbα (Nr1d1) and Dbp , which in addition to their role in regulating the molecular clock, function as transcription factors that regulate expression of clock-controlled genes  [Many crucial behaviors and biological processes are regulated in a circadian-dependent manner . The masnd Clock ,7 genes. itself) ,7.e.g., proteins, nucleic acids, and lipoproteins), referred to as pathogen-associated molecular patterns (PAMPs), are detected via innate immune recognition receptors such as Toll-like receptors (TLRs) [Alterations of circadian rhythms in the form of nightshift work or genetic abnormalities have been linked to pathologies, such as sleep disorders, depression, and increased risk of cancer –11. Lipos (TLRs) . To dates (TLRs) .The aim of this study was to determine if PAMPs other than LPS alter expression of the molecular clock.ex vivo and in vivo challenge experiments, animals were euthanized at 8:00 a.m. or 8:00 p.m., which corresponds with Zeitgeber time (ZT) 1 and 13, respectively. To assess Tlr expression levels over the daily light-dark cycle, animals were sacrificed every 4 h , over a 24-h period. Tissues were immediately collected for further processing as described below. During the study, animal care and treatment complied with National Institutes of Health policy, were in accordance with institutional guidelines, and were approved by the University of Hartford Animal Institutional Animal Care and Use committee.Eight-week-old C57BL/6J male mice (The Jackson Laboratory) were fed rodent chow ad libitum, maintained under constant environmental conditions and entrained to a 12 h light / 12 h dark cycle (light period from 7:00 a.m. to 7:00 p.m.) for 2 weeks before experiments. For the Tlr expression levels over the daily light-dark cycle, approximately 5 x 105 splenocytes were suspended in 300 μl of RLT buffer (Invitrogen) containing β-Mercaptoethanol and stored at -80°C until RNA extraction was performed as described below.Spleens were collected in RPMI 1640 (Invitrogen Life Technologies) supplemented with 10% FBS. Each spleen was homogenized in 2 ml of medium and filtered through a 40 μM nylon cell strainer (BD Biosciences). Spleens and splenocyte suspensions were kept on ice throughout the entire procedure. Immediately after splenocytes were isolated from all of the spleens, they were challenged with various PAMPs. To assess 5 cells per well were added to 12-well culture plates (Corning) and were challenged with 1 ml of medium containing heat-killed Listeria monocytogenes , poly(I:C) (250 ng/ml), lipopolysaccharide , flagellin , ssRNA40 (1.25 μg/ml), or ODN1826 . TLR agonists were purchased from InvivoGen. After the plates were incubated at 37°C in 5% CO2 for 3.5 h, the supernatant was removed, and the cells were washed with PBS. 300 μl of RLT buffer (Invitrogen) containing β-Mercaptoethanol was then added to each of the wells. Cell lysates were stored at -80°C until RNA extraction was performed as described below.Approximately 5 x 106 cells were pelleted and resuspended in 600 μl of RLT buffer (Invitrogen) containing β-Mercaptoethanol. Cell lysates were stored at -80°C until RNA extraction was performed as described below.Mice were injected intraperitoneally with 30 μg poly(I:C) (InvivoGen) or PBS at ZT1 and ZT13. Mice were sacrificed 3.5 or 48 h after challenge, in two separate experiments. Spleens were collected and cells isolated as described above. Approximately 10Actb Mm00607939_s1, Per2 Mm00478113_m1, Rev-erbα Mm00520708_m1, Bmal1 Mm00500226_m1, Dbp Mm00497539_m1, Tlr1 Mm00446095_m1, Tlr2 Mm00442346_m1, Tlr3 Mm01207404_m1, Tlr5 Mm00546288_s1, Tlr6 Mm02529782_s1.RNA from splenocytes was isolated using the RNeasy Mini kit (Qiagen) in conjunction with the RNase-Free DNase Set (Qiagen). cDNA was synthesized using the high capacity cDNA reverse transcription kit according to manufacturer’s instructions (ThermoFisher). Relative quantitation of mRNA levels was performed by quantitative PCR via TaqMan Gene Expression Assays (ThermoFisher) and TaqMan Gene Expression Master Mix (ThermoFisher) using a StepOnePlus (ThermoFisher) system. Analyses were performed using the standard curve method with β-actin as the normalizing endogenous control. Relative quantitation values were determined by dividing the relative quantity for the target gene by the relative quantity for β-actin. The following TaqMan Gene Expression Assays were used (ThermoFisher): in vitro and in vivo. One-way ANOVA with the Dunnett’s posttest was used to assess daily variations in Tlr and clock gene expression between the acrophase and other time points. Two-way ANOVA with the Tukey posttest, treating time and treatment as independent factors, was used to assess differences between means of clock gene expression after PAMP challenge versus the control . A two-tailed t test was used to assess differences between means of clock gene expression after PAMP challenge versus the control  at 48-h post challenge. All analyses were performed using Prism 7.0a (GraphPad).One-way ANOVA with the Dunnett’s posttest was used to assess differences between means of clock gene expression after PAMP challenge versus the control  ex vivo with PAMPs targeting specific TLRs. After 3.5 h, quantitative PCR was used to assess mRNA levels of core clock genes, Per2 and Bmal1, as well as ancillary genes, Rev-erbα and Dbp. After challenge with Pam3CSK4, which triggers the TLR1/2 heterodimer, expression levels of Per2 were higher in splenocytes at ZT1 (pANOVA < 0.0001) and ZT13 (pANOVA < 0.01), while Dbp levels were significantly lower at both ZT1 , Rev-erbα at ZT1 (pANOVA < 0.05), and Dbp at ZT1 and ZT13  when compared to the controls , Rev-erbα at ZT1 (pANOVA < 0.0001) and ZT13 (pANOVA < 0.001), and Dbp at ZT1 (pANOVA < 0.0001) and ZT13 (pANOVA < 0.0001) after challenge with the TLR3 ligand, poly(I:C), when compared to the controls (Rev-erbα at ZT1 (pANOVA < 0.01) and Dbp at ZT1 (pANOVA < 0.0001) and ZT13 (pANOVA < 0.0001)  and ZT13 (pANOVA < 0.0001), and lower mRNA levels were detected for Bmal1 at ZT1 (pANOVA < 0.001), Rev-erbα at ZT1 (pANOVA < 0.05), and Dbp at ZT1 (pANOVA < 0.0001) and ZT13 (pANOVA < 0.0001), when compared to the unchallenged controls  . After ccontrols . This rein vivo for one of the TLR agonists, mice were challenged intraperitoneally with either poly(I:C) or phosphate buffered saline (PBS) at ZT1 or ZT13. Splenocytes were isolated after 3.5 h, and clock mRNA levels were assessed via qPCR. In agreement with the ex vivo experiments , Rev-erbα (pANOVA < 0.0001), and Dbp (pANOVA < 0.0001) at ZT1, while no changes were detected in Per2 and Bmal1 expression at ZT13, compared to the control groups  down to the molecular level  and does so for nearly all aspects of physiology and behavior [e.g., mice, rats, and human subjects) have revealed that LPS, a PAMP recognized by TLR4, alters clock gene expression in various cell types and tissues  [The circadian clock regulates homeostasis from the organismal level  –15,17,20Dbp was the gene most affected by PAMP challenge, as its expression was significantly reduced after challenge with all the PAMPs that were assessed, except for ssRNA40 at ZT13, which led to an increase in Dbp expression  could still be significantly affected. For example, DBP has been shown to play a role in regulating locomotor activity and sleep regulation, as well as maintaining proper renal function and regulating xenobiotic metabolism [All of the PAMPs tested significantly altered expression of at least one clock gene in mouse splenocytes . Dbp waspression . Even thtabolism –24.Tlr9 expression varies throughout the day in mouse spleen, while Tlr4 and Tlr7 expression do not [Tlr1, 3, and 5, but not Tlr2, expression were observed in the spleen  challenge at ZT1, expression of Per2, Bmal1, Rev-erbα, and Dbp were all significantly lower than the controls, while significant differences in gene expression were not observed when mice were challenged at ZT13  challenges, some discrepancies were observed  except for Per2 in the ex vivo challenged, and not observing a difference at ZT13 for all genes (both in vivo and ex vivo) except for Rev-erbα and Dbp, which were significantly lower in splenocytes during the ex vivo challenge.Following an  at ZT13 . These rStreptococcus pneumoniae recognition [e.g., influenza virus), which to a certain extent could be mediated via clock alteration, could potentially predispose an individual to a secondary infection (e.g. S. pneumoniae) [In the current study, individual PAMPs were shown to alter expression of the molecular clock. However, upon pathogen infection of a host, multiple TLRs are potentially activated by different microbial components. For example, influenza virus activates TLR3, 7, and 8  and TLR2ognition –34. Therognition ,26,29, cumoniae) ,36.Here, clock expression was altered in immune cells, which directly interact with PAMPs. Further investigation is warranted to determine if these changes in clock gene expression are observed systemically, including but not limited to the SCN. One of the hallmark symptoms upon infection is the feeling of fatigue. It is intriguing to speculate that disturbances to the central clock via PAMP-TLR interaction could lead to an altered sleep state, which could be triggered in order to maximize host recovery during infection ,37. In aS1 FigMice were challenged with poly(I:C) or PBS at ZT13, relative clock mRNA levels  were determined by qPCR 48 h after challenge in splenocytes, and calculated as percentage of the maximum value. Data are mean + SEM of 5 and 6 animals per time point for the control and poly(I:C) challenge group, respectively. ns, not significantly different from the control  as per two-tailed t test.(TIF)Click here for additional data file.S2 FigPer2, Rev-erbα, and Tlr gene expression in splenocytes. Relative mRNA levels at each time point were determined by qPCR and calculated as the relative expression over the 24-h period. Data are mean ± SEM of 5 animals per time point. One-way analysis of variance (ANOVA) was used to make comparisons between the acrophase and other time points. *p < 0.05, **p < 0.01, *** p < 0.001. Open bar indicates light period, while colored bar indicates dark period.Daily variations in (TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


These colonies consisted of various cell types. Flk-1+/CD31−/CD45−/CD71−, and CD45+ and/or CD71+ cell populations produced CFU-GEMM and CFU-GM, or CFU-GM and CFU-E colonies, respectively. Immunohistochemical evaluations of smears prepared from colonies revealed the presence of cells of different hematopoietic lineages. These cells were characterized by labeling with various combinations of antibodies directed against CD31, CD41, CD71, c-kit, Mpl, Fli1, Gata-2, and Zeb1 markers. Furthermore, we found that proepicardium-specific marker WT1 co-localized with Runx1 and Zeb1 and that single endothelial cells bearing CD31 molecule expressed Runx1 in the proepicardial area of embryonic tissue sections. We have shown that cells of endothelial and/or hematopoietic phenotypes isolated from mouse proepicardium possess hematopoietic potential in vitro and in situ. These results are supported by RT-PCR analyses of proepicardial extract, which revealed the expression of mRNA for crucial regulatory factors for hemogenic endothelium specification, i.e., Runx1, Notch1, Gata2, and Sox17. Our data are in line with previous observation on hemangioblast derivation from the quail PE.During embryonic development, hematopoietic cells are present in areas of blood-vessel differentiation. These hematopoietic cells emerge from a specific subpopulation of endothelial cells called the hemogenic endothelium. We have previously found that mouse proepicardium contained its own population of endothelial cells forming a network of vascular tubules. We hypothesize that this EC population contains cells of hematopoietic potential. Therefore, we investigated an in vitro hematopoietic potential of proepicardial cell populations. The CD31The online version of this article (10.1007/s00418-018-1661-1) contains supplementary material, which is available to authorized users. During fetal life, hematopoiesis begins at 7.5 dpc (day post coitum) in blood islands located in the endodermal wall of the extraembryonic yolk sac  called the hemogenic endothelium (HE)  is a small, transient structure essential to heart development, which appears on the venous pole of murine embryonic heart from 9.0 dpc to 9.75 dpc  , a colony-forming cell (CFC) assay , and a laser confocal microscopy of immunohistochemically stained cells labeled with antibodies against different hematopoietic lineages markers. We also employed RT-PCR to assess the mRNA expression of some transcription factors crucial for the HE formation, e.g., Notch1, Gata2, Runx1, Sox17, Nkx2-5. Our study is the first to demonstrate that the mammalian PE contains specific-cell populations which in vitro exhibit hematopoietic potential as evidenced by the colony-forming cell assay and by in situ mRNA expression for genes specific for the HE.The experiments were performed with F1 cross of C57BL/6 and CBA mouse inbred strains. One hour after mating, the presence of the vaginal plug was assessed, and that day was designated as 0 dpc. Embryos were collected at 9.5 dpc (22–25 somites stage). Mice were euthanized, and after immediate excision, embryos-containing uteri were placed in a culture dish with sterile Tris-Tyrode solution. Under an inverted microscope, embryos were excised from the uteri, devoid of fetal membranes, and positioned on the left side for better visualization of the PE. Proepicardium isolation was performed according to a method described earlier  Local Ethics Committee at the Medical University of Warsaw, Poland.2+- and Mg2+-free PBS  and digested with accutase  on a magnetic stirrer at room temperature for 15 min. PE tissue fragments were intensively pipetted, and if still not fully dissociated, digested for additional 10 min with accutase. Isolated cells were washed in a PBS solution containing 10% FCS , and then transferred through a nylon filter membrane with pore size of 40 µm. Using a magnetic-activated cell sorting system , cells were separated into different populations with indirect magnetic labeling, following the manufacturer’s instructions. In brief, the cells were first incubated with primary rat antibodies against CD31 , CD71 , CD45 , and Flk1 . Next, they were incubated with secondary anti-rat antibodies conjugated with microbead . After labeling with the antibodies, the cells were separated with MACS Columns and Separators. First, the cells were subdivided into two populations: CD45/CD71 double-positive, and CD45/CD71 double-negative. Subsequently, CD31-positive and CD31-negative cells were isolated from the latter population. Finally, Flk-1-positive cells were separated from CD31-negative cells. In this way, the following cell populations were obtained and used for further procedures: Flk-1+/CD31−/CD45−/CD71−; Flk-1−/CD31−/CD45−/CD71−; CD31+/CD45−/CD71−; and CD45+ and/or CD71+. These steps in cell subpopulations isolation are shown in Scheme The proepicardia were washed with Ca™ GF M3434  on 24-well culture plates. No additional exogenous cytokines were added to the culture medium. Cultures were maintained in an incubator in a controlled atmosphere with a temperature of 37 °C, 5% CO2 concentration, and 95% relative humidity. The colonies were identified using an inverted microscope, and inspected at day 3, 5, and 7. At day 10, different types of colonies growing in the culture were counted, and the cells from these colonies were collected for smear preparation. Those cells were either stained with Giemsa, or immunostained with antibodies directed against a variety of antigens specific for hematopoiesis.The separated cell populations were suspended in Iscove’s MDM with 2% FBS and cultured in Methocult®, ThermoFisher Scientific, MA, USA) at room temperature for 20 min. Next, the smears were washed with PBS and incubated with 1% BSA, 0.1% TritonX-100, and 0.1 glycine in PBS for 30 min. Then, for 1 h, the smears were labeled with a mixture of two or three primary antibodies, directed against different antigens, diluted in PBS containing 1% BSA. Primary antibodies used for labeling are listed in Table The collected cell smears were dried for 30 min at 36 °C, and then fixed with 4% buffered paraformaldehyde  in PBS . Immunohistochemical negative control sections were prepared using the same, previously described procedure, except that the primary antibodies were missing and replaced by PBS with 1% BSA.Subsequently, after three washes in PBS, the cells were incubated with secondary donkey antibodies at room temperature for 1 h. Then, the smears were washed in PBS. The following secondary antibodies conjugates were used: Cy3™: HeNe 543 laser 94%, PMT2 detector, smart offset − 40%, pinhole 152, smart gain 1033, filter—TD488/543/633; FITC: Argon laser 94%, PMT3 detector, smart offset − 40%, pinhole 148, smart gain 1028, filter—TD488/543/633; C: Dylight™: HeNe 633 laser 37%, PMT3 detector, smart offset − 40%, pinhole 132, smart gain 962, filter—TD488/543/633.Next, to visualize cell nuclei, the smears were stained with DAPI  at room temperature for 2 min, and then mounted with DAKO immunofluorescent medium . A Leica confocal microscope-type TCS SP5 was used for cell smears analysis. Basic system settings were: objective: Leica HC PL APO ×20/0.7; fluorescence settings for DAPI: UV 405 diode 12%, PMT1 detector, smart offset − 40%, pinhole 132, smart gain 1250, filter—substrate; Cy3First, the smears were fixed in 100% methanol for 30 min, next rinsed in tap water, and then treated with a freshly made solution of 10% Giemsa stain  for 30 min.Immediately after collecting, the 9.5 dpc embryos were fixed in 4% buffered paraformaldehyde for 30 min and processed through 10–20–30% sucrose. Then, they were embedded in Tissue Freezing Medium  and frozen in liquid nitrogen. The frozen embryos were cut serially into 10 µm sections. The sections were washed with PBS, and subsequently incubated with 1% BSA, 0.1% TritonX-100, and 0.1 glycine in PBS for 30 min. After the incubation, the sections were blocked with 5% donkey serum  for 1 h. The following primary antibodies, diluted in PBS/1% BSA, were used for labeling at room temperature for 1 h: anti-WT1, anti-CD31, anti-Runx1, and anti-Zeb1  according to the producer’s protocol. The quality and concentration of RNA were assessed with NanoDrop spectrophotometer. The RT was performed with High Capacity RNA-to-cDNA Kit, according to the producer’s protocol . The cDNA was stored at − 20 °C. Gene expression was measured with the relative quantitation (RQ) using comparative CT assay (Livak and Schmittgen PE (9.5 dpc) and liver tissue fragments (13.5 dpc) were used for RNA isolation. RNA was isolated with NucleoSpinThe statistical analysis was performed with Statistica software ver. 12 . Descriptive data are presented as means and standard deviations obtained from six experiments. To compare parameters between groups, the Mann–Whitney–Wilcoxon test was used, with the level of significance set at 0.05.+/CD45−/CD71− cell population. These colonies were of different types, such as: CFU-GEMM .A lower number of colonies were obtained with cells of Flk1−/CD31−/CD45−/CD71−, did not form any colonies.Quadruple-negative cells, Flk1Giemsa-stained cellular smears confirmed the classification of different types of colonies, based on their morphology Fig. d.+/CD45−/CD71− cell fraction) revealed a heterogeneous cell population: numerous CD41+/CD31+ cells, and some CD41+ /CD31− or CD41− /CD31− cells  or megakaryocytic (Fli1+) lineages, were observed, too , was observed in cells expressing or not expressing the c-kit molecule (a tyrosine kinase receptor expressed in hematopoietic stem cells and progenitor cells). However, it must be pointed out that some c-kitpl− Fig. a–d.+/Zeb1−, c-kit−/Zeb1+, and c-kit−/Zeb1− cells were observed as well. Some of the c-kit+ cells were also positive for Notch1 marker. However, only some of those double-positive cells (c-kit+/Notch1+) also co-expressed Zeb1 . Expression of these two markers varied in cells, as c-kiteb1 Fig. a–f.+/CD31−/CD45−/CD71− cell population.Similar cell phenotypes were observed in cell smears from CFU-GEMM colonies, which were derived from Flk1+/CD45−/CD71−, Flk1+/CD31−/CD45−/CD71−, and CD45+ and/or CD71+ cell populations. Cells from this type of colonies were characterized by various combinations of CD41 and/or Gata2 expression: CD41+/Gata2+, CD41+/Gata2−, CD41−/Gata2+, and CD41−/Gata2−  and megakaryocytic (Fli1+) lineages  or single-positive  cells were present. This phenomenon may be interpreted as a sign that these cells were at different stages of differentiation, since erythrocytes and megakaryocytes arise from a common progenitor  the latter being detected in the proepicardial organ at developmental stage in which PE had contacted the heart, these authors speculated that both cell types derived from the same structure, namely, PE. On the other hand, our previous study demonstrated some CD45+ cells expressing the Lyve1 antigen located in the subepicardium of mouse heart what suggests their macrophage identity  . It cannot be excluded that Runx1+/WT1+/CD31− cells play a role in coronary plexus formation after differentiating into the epicardium and EPDCs. It has been reported that Runx1 is required for the proper formation of coronary vessels during cardiogenesis, and that Runx1-null mice have an underdeveloped coronary plexus in comparison with the wild-type mice  assay, and by in situ immunohistochemical labeling of cell lineages cultured in colonies. We also found that mouse PE cells express mRNA for Runx1, Notch1, Gata2, Sox17, and Nkx2-5, which are the key factors involved in activity of hemogenic endothelium.In conclusion, our major and novel finding is that cells isolated by us from the mammalian (mouse) PE and bearing the CD31Below is the link to the electronic supplementary material.Supplementary material 1 (TIF 7343 KB)Supplementary material 2 (TIF 7369 KB)