Here, we examine known GTPase regulators of vesicle trafficking events to assess whether they affect endothelial cell (EC) lumen and tube formation. We identify novel roles for the small GTPases Rab3A, Rab3B, Rab8A, Rab11A, Rab27A, RalA, RalB and caveolin-1 in co-regulating membrane trafficking events that control EC lumen and tube formation. siRNA suppression of individual GTPases such as Rab3A, Rab8A, and RalB markedly inhibit tubulogenesis, while greater blockade is observed with combinations of siRNAs such as Rab3A and Rab3B, Rab8A and Rab11A, and RalA and RalB. These combinations of siRNAs also disrupt very early events in lumen formation including the formation of intracellular vacuoles. In contrast, knockdown of the endocytosis regulator, Rab5A, fails to inhibit EC tube formation. Confocal microscopy and real-time videos reveal that caveolin-1 strongly labels intracellular vacuoles and localizes to the EC apical surface as they fuse to form the luminal membrane. In contrast, Cdc42 and Rab11A localize to a perinuclear, subapical region where intracellular vacuoles accumulate and fuse during lumen formation. Our new data demonstrates that EC tubulogenesis is coordinated by a series of small GTPases to control polarized membrane trafficking events to generate, deliver, and fuse caveolin-1-labeled vacuoles to create the apical membrane surface. Critical steps during early vasculogenesis and endothelial cell (EC) lumen formation include both the establishment of asymmetric cytoskeletal polarization  and the trafficking of pinocytic intracellular vacuoles or other intracellular vesicles to create an apical membrane surface–16. Impoin vitro and in vivo[ECs and other cell types are known to transport membrane compartments through endocytic mechanisms including clathrin-dependent, clathrin-independent, caveolae/caveolin-1 dependent, as well as macropinocytic and phagocytic mechanisms–26. Membd in vivo, 39. Addd in vivo. TogetheIn this study, we present novel information characterizing the role of regulators of membrane trafficking events during EC lumen formation and tubulogenesis. Our work has identified new roles for the GTPases Rab3A, Rab3B, Rab8A, Rab11A, Rab27A, RalA and RalB as well as Caveolin-1 during this process and siRNA suppression of individual and combinations of these GTPases markedly impairs the ability of ECs to undergo both vacuolation and tubulogenesis. Furthermore, we show that intracellular vacuoles are highly enriched in Caveolin-1, Src family kinases and RalA which then proceed to target to the apical membrane surface while the small GTPases Cdc42, Rab11A and Rab27A are localized in a polarized, subapical region. Thus, EC lumen and tube morphogenesis are controlled by sequential events regulating intracellular vacuole generation, polarized vesicle trafficking and fusion that occurs in coordination with key small GTPases, signaling pathways and cytoskeletal polarization to control how ECs form cell-lined tube networks in 3D extracellular matrix environments.O-tetradecanoyl-phorbol-13-acetate (TPA) were obtained from Sigma-Aldrich . MBQ-167 was obtained from Selleckchem . Recombinant fibroblast growth factor 2 (FGF-2), antibody against β-Actin and the chemical inhibitors PP2 and GM6001 were obtained from EMD Millipore . Antibodies against Rab3B, Rab3D, Rab11A and Rab27A were obtained from Abcam . Antibodies against Caveolin-1, Rab3A, Rab5 and Rab8 were obtained from Cell Signaling Technology . An antibody against RalA was obtained from BD Biosciences . GFP-RalA and S-Ch-Cdc42 adenoviruses were generated and utilized as described previously[Stem cell factor (SCF), stromal cell-derived factor 1 alpha (SDF-1α) and interleukin-3 (IL-3) were obtained from R&D systems . Chemical inhibitors PP3, EHT 1864, and Nexinhib20 were obtained from Tocris . Ascorbic acid and 12-reviously.Human umbilical vein endothelial cells were obtained from Lonza  and were cultured (passage 3–6) as described previously.6 cells/mL in 2.5 mg/mL collagen type I matrices and assays were performed as described previously[For use in siRNA-mediated knockdown assays, siRNA transfected ECs were harvested and then suspended at 2 x 10reviously, 41. In 6 cells/mL in 3.75 mg/mL collagen type I matrices and assays were performed as described previously[For use in confocal microscopy imaging analysis, ECs were first infected with recombinant adenoviruses as described previously. Infectereviously. In briereviously. ECs asssiRNA protocols using the siRNA list below were performed as described previously. ECs wersiRNAs from Ambion are as follows:Control (AM4637) Silencer Select Negative Control #25’-CCAUCACCACCGCAUACUA-3’Rab3A (s11666) 5’-GCUUCAUUCUGAUGUAUGA-3’Rab3B (s11669) 5’-AGGAGAACAUCAAUGUGAA-3’Rab3D (s18326) 5’-GGAAGAGGAGUAGACCUUA-3’Rab5A (s11678) 5’-GCAAGAGAAUUAAACUGCA-3’Rab8A (s8679) 5’-GAGAUUUACCGCAUUGUUU-3’Rab11A (s16703) 5’-GCCUCUACGGAUCAGUUAA-3’Rab27A (s11693) 5’-GGACUACGCUGCAAUUAGA-3’RalA (s11758) 5’-CAUGAAUCCUUUACAGCAA-3’RalB (s11762) 5’-GCUUCCUGAUUGAGAUUCA-3’Caveolin-1 (s2446) EcoRI-HF and BamHI-HF and XhoI and AgeI-HF restriction enzymes respectively . Amplified S-epitope (S) tagged S-Ch-Rab5A, S-Ch-Rab11A, S-Ch-Rab27A, Src-GFP-S and sfGFP-Caveolin1 constructs were subcloned into pShuttle-CMV expression plasmid using NotI-HF and XbaI, SalI-HF and NotI-HF and KpnI-HF and NotI-HF restriction enzymes respectively (New England Biolabs). Recombinant adenoviral vectors were then generated and propagated as previously described[Rab11A and Rab27A were amplified from cDNA obtained from Missouri S&T cDNA Resource center  and mCherry-Rab5A and sfGFP-Caveolin1 fusion constructs were amplified from cDNA obtained from Addgene  and Src was amplified from cDNA obtained from Open Biosystems . Standard restriction digest cloning protocol was used to subclone amplified Rab11A and Rab27A into pmCherry-C1 plasmid and Src into pAcGFP-N1 plasmid  using described. The PCR5’-AGGAATTCTATGGGCACCCGCGACGACGAGTAC-3’Rab11A 5’-AGGGATCCTTAGATGTTCTGACAGCACTGCAC-3’5’-AGGAATTCTATGTCTGATGGAGATTATGATTACC-3’Rab27A 5’-AGGGATCCTCAACAGCCACATGCCCCTTTCTCC-3’5’-AGCTCGAGATGGGTAGCAACAAG-3’Src 5’-AGACCGGTATGAGGTTCTCCCCG-3’Primers used for subcloning into the pShuttle-CMV plasmid are as follows. The first primer (S-Cherry) is an upstream primer followed by downstream primers for S-Ch-Rab5A, S-Ch-Rab11A and S-Ch-Rab27A. The following sets are pairs of upstream and downstream primers for Src-GFP-S and sfGFP-Caveolin1 respectively.5’-AGGCGGCCGCACCATGGCAAAAGAAACCGCTGCTGCGAAATTTGS-Cherry AACGCCAGCACATGGACTCGATGGTGAGCAAGGGCGAGGAG-3’5’-AGTCTAGATTAGTTACTACAACACTGATTCCTGGTTGGC-3’Rab5A 5’-AGTCTAGATTAGATGTTCTGACAGCACTGCAC-3’Rab11A 5’-AGTCTAGATCAACAGCCACATGCCCCTTTCTCC-3’Rab27A 5’-AGGTCGACATGGGTAGCAACAAGAG-3’Src-GFP-S 5’- AGGCGGCCGCTTACGAGTCCATGTGCTGGCGTTCAAATTTCGCAGCAGCGGTTTCTTTACCAGACTTGTACAGCTCATCCATGCCGTG-3’5’-AGGGTACCACCATGGTGAGCAAGGGCGAGGAGCTGTTC-3’sfGFP-Caveolin1 5’-AGGCGGCCGCTTATATTTCTTTCTGCAAGTTGATGCGG-3’Fluorescent imaging of 3D EC cultures undergoing tube formation was performed using a confocal microscope  with excitation wavelengths of 405 and 488 or 405, 488 and 561 nm sequentially. We used a 63X water immersion objective (NA 1.2) to capture high-resolution images and in addition, Leica application suite advanced fluorescence (LAS-AF) software was utilized to obtain and process images. 3D endothelial cultures were stained with toluidine blue and imaged using light microscopy and inverted microscopes . EC culture photographs were analyzed using Metamorph software  that allowed for the quantification of vessel and lumen areas.Statistical analysis of selected EC vasculogenic and lumen formation data was performed using Microsoft Excel (Microsoft) or Prism 8 (GraphPad). Statistical significance was set at p< 0.05. Student’s t-tests were used when analyzing two groups within a single experiment (with a minimum n = 10 from representative experiments or consolidated experiments and a range from n = 10 to n = 36). In all cases, each experimental condition was performed in triplicate and data points were derived from representative images from each of these wells. ANOVA with post-hoc Tukey analysis was performed when multiple comparisons between samples were evaluated.Fig 1). Our data implicates a key role for Rab3A, Rab3B, Rab8A, Rab11A, Rab27A and Caveolin-1 during this process where Rab3A and Rab8A knockdown have the most marked effects. In contrast, Rab5A knockdown showed no effects whereas Rab3D may be inhibitory as knockdown stimulated EC tubulogenesis . Using combinations of siRNAs, we demonstrated that combined knockdown of Rab8A with Rab11A or Rab27A had a greater blocking effect compared to knockdown of Rab8A alone and that combined knockdown of Rab27A and Caveolin-1 had a greater blocking influence than knockdown of Rab27A or Caveolin-1 alone. Furthermore, these combined knockdowns had the greatest blocking effect compared to other siRNA combinations tested . This data suggests that vesicle trafficking events controlled by Rab3A, Rab3B, Rab8A, Rab11A, Rab27A and Caveolin-1 appears to be necessary for ECs to form lumens and assemble into capillary networks in 3D matrix environments whereas, the endosomal trafficking regulator, Rab5A, does not appear to be involved.Our previous work suggests a key role for small GTPases such as Cdc42, Rac1, Rac2, k-Ras, and Rap1b, and their downstream effectors such as Pak2, Pak4 and Rasip-1, in controlling EC lumen formation, 16, 43.Fig 1), we addressed the role of both isoforms of the Ral GTPases, which are known to regulate exocytosis through their association with the exocyst complex[Fig 4). Using additional combinations of siRNAs, we show that combined knockdown of RalB with Rab3A, Rab3B, Rab27A, and RalA had the greatest blocking effects compared to any of the blocking effects observed with individual siRNAs . We also show that combined siRNA suppression of Rab3A and Rab8A has a greater blocking effect than suppression of Rab3A alone . Together, this data suggests that RalA and RalB are critical regulators of EC lumen morphogenesis. The combined influence of RalB siRNA knockdown with other GTPases associated with the regulation of exocytosis also supports the idea that these molecules play key functional roles during lumen formation. Furthermore, our results from combined siRNA suppression of Rab3A and Rab8A demonstrates a possible role for regulation of differing endocytic and exocytic membrane vesicle trafficking steps during EC tubulogenesis.Because we observed strong blocking effects on tubulogenesis with siRNA suppression of Rab3A, Rab3B and Rab27A  , and these siRNAs also similarly blocked intracellular vacuole formation . We anticipated that there would be distinctions between them, but there was not, demonstrating that these different classes of vesicle trafficking regulators, have overlapping functions in regulating both early and later steps in the lumen formation process.Our previous work demonstrates a major role for intracellular vacuole formation as a key initiation step in stimulating the EC lumen formation process. To address whether we can detect differences in the temporal role of vesicle trafficking regulators vs. previously identified regulators of EC intracellular vacuole and lumen formation, we performed siRNA suppression and pharmacologic inhibition experiments. We focused on combinations of siRNAs that had strong inhibiting effects on lumen formation . These results are similar to that observed with siRNA combinations including Rab27A siRNA . In addition, we compared these results to addition of Cdc42 and Rac inhibitors such as MBQ-167 (Cdc42 and Rac inhibitor), and EHT1864 (Rac inhibitor) which interfere with both EC intracellular vacuole and lumen formation . Interestingly, an inhibitor of MT1-MMP, GM6001, strongly blocks lumen formation, but has less influence on intracellular vacuole formation . Our data does suggest that lumen expansion requires MT1-MMP and this appears to require intracellular vacuole-vacuole fusion events. We observe retention of EC intracellular vacuoles (labeled with GFP-caveolin-1) during lumen formation in the presence of GM6001, particularly observed later in the process . Initial vacuole formation is not inhibited by GM6001, but further lumen formation is markedly inhibited, although vacuoles are retained over time in the GM6001-treated ECs, but not in control ECs. In contrast, addition of the lumen formation inhibitors, PP2 (Src inhibitor) and EHT1864, markedly block both vacuole and lumen formation, while PP3, an inactive control for PP2, has no effect . Overall, these data indicate a complex and overlapping role for Rab and Ral GTPases in controlling both early (through intracellular vacuole formation) and later phases of lumen formation (vacuole-vacuole fusion and lumen expansion events).We also tested pharmacologic inhibitors for a comparison, and interestingly, the Rab27A inhibitor, Nexinhib20, blocked lumen formation, but also intracellular vacuole formation at different early time points during EC lumen formation (S5 Videos) and confocal microscopic imaging over time . Here, by using sfGFP-Caveolin-1 and GFP-RalA labeled ECs, we show that Caveolin-1  and RalA  strongly localize to intracellular vacuoles generated from the basal membrane surface that are trafficked apically and fuse within a polarized perinuclear region to form the EC apical luminal membrane over time. Real-time videos demonstrate marked targeting of sfGFP-Caveolin-1 and GFP-RalA to intracellular vacuoles and the developing apical surface, while S-mCherry (S-Ch)-Rab11A, S-Ch-Cdc42 and S-Ch-Rab5A did not (S5 Videos). Additionally, using a Src-GFP-S construct we show that Src also targets to intracellular vacuoles during this process . As the intracellular vacuoles coalesce to form the lumen during later events, we also show that Caveolin-1, RalA, and Src target to the apical membrane surface. In contrast, use of S-Ch-Cdc42, S-Ch-Rab11A, S-Ch-Rab27A and S-Ch-Rab5A constructs showed that Cdc42, Rab11A, Rab27A and Rab5A all show targeting ability to the subapical domain region . During early EC lumen formation, Rab11A appears to localize to vesicles in a punctate manner that accumulate in a polarized, perinuclear region  whereas Rab27A is initially diffuse throughout the cytoplasm  and Rab5A is localized to more diffuse, punctate regions . Furthermore, it appears that caveolin-1-, RalA-, and Src-strongly label intracellular vacuoles which are trafficked apically where they accumulate in these polarized, perinuclear regions, that are enriched in Rab11A and Cdc42.To investigate the subcellular localization of additional key regulators of EC lumen formation and molecules associated with membrane trafficking events, we generated a panel of recombinant adenovirus reagents expressing mCherry-fusion or GFP-fusion proteins. We then used multiple combinations of these reagents to assess whether these molecules co-localize with one another over time during different stages of EC lumen morphogenesis as assessed using real-time movies . We also demonstrate critical roles for key molecular regulators of EC tubulogenesis in controlling apical-basal polarization such as Caveolin1, RalA and Src which markedly target to intracellular vacuoles and the developing apical membrane, while Cdc42, Rab11A and Rab27A localize within a subapical domain. Finally, we demonstrate that generation of intracellular vacuoles is controlled by multiple GTPases including Rab27A, Rab3A, Rab11A, RalA and RalB in addition to Cdc42, Rac, and Src activities which are necessary for EC lumen formation. Interestingly, MT1-MMP, which has much less influence on intracellular vacuole formation appears to have a key downstream function in facilitating vacuole-vacuole fusion events to generate and expand the luminal compartment.Intracellular vacuolization is a critical mechanism controlling EC lumen, tube and capillary assembly, 19, 46.de novo lumen formation. Recent work in Madin-Darby Canine Kidney cyst models of epithelial lumen formation have implicated roles for Rab11A-Rab8A and Rab27/Rab3/Rab8 pathways in controlling de novo development of lumen cysts[As Rab GTPases are the largest family of small GTPases and regulate a variety of membrane trafficking events, 28, 30,men cysts, 31, 32.men cysts, 45, 47.men cysts. Our stuThe work presented here identifies new regulators of EC polarity and trafficking of intracellular vacuoles during tubulogenesis. We show that as lumen formation progresses, ECs accumulate intracellular vacuoles generated from the basal membrane surface that are enriched in Caveolin-1, RalA, and Src. Of great interest are previous studies implicating a co-role for these molecules in regulating vesicular trafficking events, inducing membrane curvature in developing vesicles, and activation of caveolae to regulate membrane transport functions in ECs and other cell types, 48–50. Overall, the work presented here advances our understanding of key regulators of EC tubulogenesis and adds that key vesicle trafficking regulators of the Rab and Ral family coordinate with other key small GTPases including Cdc42, Rac1, Rac2, k-Ras, and Rap1b and their effectors to control this critical and complex process. These new studies further support our longstanding work demonstrating a critical role for intracellular vacuole formation in the EC lumen formation process. In a coordinated manner, these key small GTPases impact the formation of intracellular vacuoles, vacuole-vacuole fusion events, the polarization of these processes within individual ECs via the apical membrane and underlying modified microtubule cytoskeleton, and maturation of the EC apical membrane to create multicellular networks of EC-lined tubes.S1 FigControl and siRNA knockdown samples for each molecule were loaded side-by-side and in between colored molecular weight markers. Blots were cut along the molecular weight marker lanes and were probed with the indicated antibodies and then secondary antibodies coupled to horseradish peroxidase followed by development with enhanced chemiluminescence reagents. Blots were lined up together and exposed to x-ray film. (A) Two independent knockdown experiments are shown together for each of the indicated molecules. (B) The same blots shown in A were stripped and then probed with anti-actin antibodies.(TIF)Click here for additional data file.S2 FigControl and siRNA knockdown samples for each molecule were loaded side-by-side and in between colored molecular weight markers. Blots were cut along the molecular weight marker lanes and were probed with the indicated antibodies and then secondary antibodies coupled to horseradish peroxidase followed by development with enhanced chemiluminescence reagents. Blots were lined up together and exposed to x-ray film. (A) A representative knockdown experiment is shown for each of the indicated molecules. The upper panel and lower panels show different exposures of the same blot. (B) The same blots shown in A were stripped and then probed with anti-actin antibodies. The upper panel and lower panels show different exposures of the same blot.(C) A representative knockdown experiment is shown for RalA. The same blot was stripped and probed with anti-actin antibodies.(TIF)Click here for additional data file.S1 VideoECs were induced to express sfGFP-caveolin-1 and mCherry-Rab11A and then were seeded in 3D collagen matrices for 24 hr. Large arrows indicate key areas to focus on in the video, while small arrows indicate the developing apical membrane surface. The video is shown at 8 frames/sec.(MP4)Click here for additional data file.S2 VideoECs were induced to express GFP-RalA and mCherry-Rab11A and then were seeded in 3D collagen matrices for 24 hr. Large arrows indicate key areas to focus on in the video, while small arrows indicate the developing apical membrane surface. The video is shown at 8 frames/sec.(MP4)Click here for additional data file.S3 VideoECs were induced to express sfGFP-caveolin-1 and mCherry-Rab11A and then were seeded in 3D collagen matrices for 24 hr. Large arrows indicate key areas to focus on in the video, while small arrows indicate the developing apical membrane surface. The video is shown at 8 frames/sec.(MP4)Click here for additional data file.S4 VideoECs were induced to express GFP-RalA and mCherry-Rab11A and then were seeded in 3D collagen matrices for 24 hr. Large arrows indicate key areas to focus on in the video, while small arrows indicate the developing apical membrane surface. The video is shown at 8 frames/sec.(MP4)Click here for additional data file.S5 VideoECs were induced to express sfGFP-caveolin-1 and mCherry-Rab5A and then were seeded in 3D collagen matrices for 24 hr. Large arrows indicate key areas to focus on in the video, while small arrows indicate the developing apical membrane surface. The video is shown at 8 frames/sec.(MP4)Click here for additional data file.


I) index (10.48 for each) were selected for further analysis. Their 3D structures were predicted using I-TASSER and PEP-FOLD servers while ProSA, PROCHECK, and ANOLEA were used to validate them. The models predicted by I-TASSER were found to be better than those predicted by PEP-FOLD upon validation. Two I-TASSER models with the lowest c-score of −0.10 and −0.30 for A15_B and A15_E peptide-motifs, respectively, were selected for docking against known bacterial-antimicrobial target-proteins retrieved from protein databank (PDB). Carbapenam-3-carboxylate synthase  yielded the lowest docking energy  against motif A15_B and A15_E, respectively, using AutoDock VINA. Further, in addition to Carbapenam-3-carboxylate synthase, these peptides (A15_B and A15_E) were found to as well bind to membrane protein (PDB ID: 1by3) and Carbapenem synthetase (PDB: 1q15) when ClusPro and HPEPDOCK tools were used. The membrane protein yielded docking energy scores (DES): −290.094, −270.751; coefficient weight (CW): −763.6, 763.3 for A15_B and A15_E) whereas, Carbapenem synthetase (PDB: 1q15) had a DES of −236.802, −262.75 and a CW of −819.7, −829.7 for peptides A15_B and A15_E, respectively. Motif A15_B of amino acid positions 2–19 in Pleurocidin exhibited the strongest in silico antimicrobial potentials. This segment could be a good biological candidate of great application in pharmaceutical industries as an antimicrobial drug candidate.Antimicrobial resistance remains a great threat to global health. In response to the World Health Organizations’ global call for action, nature has been explored for novel and safe antimicrobial candidates. To date, fish have gained recognition as potential source of safe, broad spectrum and effective antimicrobial therapeutics. The use of computational methods to design antimicrobial candidates of industrial application has however, been lagging behind. To fill the gap and contribute to the current fish-derived antimicrobial peptide repertoire, this study used Support Vector Machines algorithm to fish out fish-antimicrobial peptide-motif candidates encrypted in 127 peptides submitted at the Antimicrobial Peptide Database (APD3), steered by their physico-chemical characteristics . The best two novel antimicrobial peptide-motifs  with the lowest instability index  and highest isoelectric point (p Infections caused by drug resistant bacteria remain one of the leading causes of death worldwide , as the Acinetobacter baumanni, Pseudomonas aeruginosa, and Klebsiella pneumoniae as the last line antibiotics   sequences, a total of 24 naturally occurring peptides (<100 amino acid residues) of fish origin , with weh, 2019) .FASTA file format were subjected to web-based Support Vector Machines (SVMs) algorithm based tool of Collection of Anti-Microbial Peptides (CAMPR3) server 1 1 . The gen, 2019)1 . The cho, 2019)1 . Helicalurn size , so as turn size . Further4  were then analyzed for their quality. Validation of these peptides structure was carried out in three phases; First by using Protein Structure Analysis (ProSA) web-server7 . In the second phase, PROCHECK was then used to measure the stereo-chemical properties of the modeled peptide-motifs  in each molecule (Due to the shortness of the peptide sequences (<30 amino acids) coupled with the absence of their experimentally attained structure for templates, the three dimensional structure of putative peptide-motifs were predicted using the Iterative Threading Assembly Refinement (I-TASSER) server4 . The pepexercise . The besLD v3.56 . Briefly-server7  which pre-motifs , and finmolecule .DrugBank database  library.To identify the most probable target-proteins of the motifs, all the approved antibiotic targets in the database  at https9  greater than 0.3   , net chaa scale) , from whThe I-TASSER modeling returned five models for each modeled peptide motif (A15_B and A15_E),while the PEP-FOLD prediction returned 10 models. The best I-TASSER models had a negative c-score. I-TASSER Model-1 for both peptides (A15_B and A15_E) had the best c-score of -0.10 and -0.03, respectively . On the z-score of -1.5, -1.27 against A15_B and A15_E, while a z-score of -1.44, -1.5 were observed for PEP-FOLD A_15_B and A_15_E models, respectively. All model z-scores were in the same range with the z-score of experimentally validated proteins, thus considered to be accurate. Likewise, ANOLEA showed that majority of I-TASSER models  had amino acid residues of the peptide chain in a favorable energy environment (with low energy-scores)   while PEDrugBank database, out of which 18 had experimentally determined structures deposited at PDB ; −8.20, −7.60 Kcal/mol), Beta-hexosaminidase protein , membrane protein , and carbapenam protein  against peptide-motif A15_B and A15_E, respectively  were able to bind with low docking energies  indicating their fairly high affinity with the selected antimicrobial target protein . The besectively . The affectively  in all tPseudomonas aeruginosa (The present study demonstrates that an online Support Vector Machines (SVMs) algorithm effectively localizes motifs of potentially best antimicrobial activity within a peptide. This technique is vital in enhancing the antimicrobial activity of peptides especially on resistant strains including ruginosa . The strin silico antimicrobial probability of 0.982 (A15_B and A15_E) were both from Pleurocidin; an AMP secreted by flatfish, Pleuronectes americanus that largely inhabits soft muddy to moderately hard bottoms of marine waters. Even so, motif A15_B proved to be much more stable (instability index −28.25), rendering it the best fragment designed. When docked with AutoDock VINA, A15_B continued as the best designed peptide motif yielding the highest binding energy  and highest number of hydrogen bond interactions (3) on Carbapenam-3-carboxylate synthase target. This indicates the motif (A15_B) binds spontaneously onto Carbapenam-3-carboxylate synthase target without consuming energy  of amino acid positions 2-19 in Pleurocidin secreted by a winter flounder fish, The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.HO, SO, and CN designed and implemented the study. JA, CA, HI, FF, JN, CK, CB, PO, HO, AM, JG, and KK performed the experiments and data analysis. All authors participated in writing and proofreading the manuscript and approved the final manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Among the many immunosuppressive cells in the tumor microenvironment, tumor-associated-macrophages (TAMs) are well known to contribute to tumor development. TAMs can be conditioned (polarized) to transition between classical M1-like macrophages, or alternatively to M2-like macrophages. Both are regulated by signaling molecules in the microenvironment. M1-like TAMs can secrete classic inflammatory cytokines that kill tumors by promoting tumor cell necrosis and immune cell infiltration into the tumor microenvironment. In contrast, M2-like TAMs exhibit powerful tumor-promoting functions, including degradation of tumor extracellular matrix, destruction of basement membrane, promotion of angiogenesis, and recruitment of immunosuppressor cells, all of which further promote tumor progression and distal metastasis. Therefore, remodeling the tumor microenvironment by reversing the TAM phenotype will be favorable for tumor therapy, especially immunotherapy.Methods: PLGA nanoparticles encapsulating baicalin and melanoma antigen Hgp peptide fragment 25-33 were fabricated using the ultrasonic double-emulsion technique. The nanoparticles were further loaded with CpG fragments and used conjugated M2pep and α-pep peptides on their surfaces to produce novel nano-complexes. The capability to target M2-like TAMs and anti-tumor immunotherapy effects of nano-complexes were evaluated by flow cytometry and confocal microscopy in vitro. We also investigated the survival and histopathology of murine melanoma models administrated with different nanocomplexes. Improvements in the tumor microenvironment for immune attack of melanoma-bearing mice were also assessed.Results: The nano-complexes were effectively ingested by M2-like TAMs in vitro and in vivo, and the acidic lysosomal environment triggered the disintegration of polydopamine from the nanoparticle surface, which resulted in the release of the payloads. The released CpG played an important role in transforming the M2-like TAMs into the M1-like phenotype that further secreted inflammatory cytokines. The reversal of TAM released cytokines and gradually suppressed tumor angiogenesis, permitting the remodeling of the tumor microenvironment. Moreover, the activated TAMs also presented antigen to T cells, which further stimulated the antitumor immune response that inhibited tumor metastasis. Activated T cells released cytokines, which stimulated NK cell infiltration and directly resulted in killing tumor cells. The baicalin released by M1-like TAMs also killed tumor cells.Conclusion: The nano-complexes facilitated baicalin, antigen, and immunostimulant delivery to M2-like TAMs, which polarized and reversed the M2-like TAM phenotype and remodeled the tumor microenvironment to allow killing of tumor cells. Malignant tumors are the major killers currently threatening human health around the world with rising morbidity and mortality Scutellaria baicalensis Georgi exhibits dual functions of immune-modulation and select cytotoxicity  et al. reported that baicalin stimulates T and B cell proliferation, independently and cooperatively with concanavalin A (Con-A) or lipopolysaccharide (LPS) et al. determined that the immune system modulation by baicalin in a patient with acute lymphocytic leukemia was due to increased production of IFN-γ in peripheral blood leukocytes Most previous studies have used CpG-ODN as a common immune stimulator to transform the TAMs phenotype. Consequently, immunological responses of T helper cells (Th1) were enhanced by IL-12, IFN-γ, and TNF-α secreted by TAMs The main priority for reversing the TAMs phenotype is to effectively transport the immune stimulator to the affected TAMs. Therefore, it is necessary to develop a delivery system with high affinity target ligands to ensure effective distribution of the immune-stimulator to specific cells. Recently, some studies have reported that a peptide, designated as M2pep (an M2-like macrophage binding peptide), as well as another α-peptide (a scavenger receptor B type 1 (SR-B1) targeting peptide), possess greater specificity to M2-like TAMs than to other leukocytes 25-33) was simultaneously encapsulated in the PLGA nanoparticles, while CpG-ODN was adsorbed to a polydopamine (pD) coating layer on the nanoparticle surface. Polydopamine (pD)  adheres to most material surfaces and permits conjugation of proteins and nucleic acids via a Michael addition or Schiff base reaction to its catechol functionalities in vitro and in vivo using this collaborative strategy to remodel the tumor microenvironment and influence tumor therapy utilizing nano-complexes to target M2-like TAMs.In this study, we successfully synthesized dual-targeting, M2-like TAMs nano-complexes by loading the anti-tumor drug and an immunostimulant to remodel the tumor microenvironment, as shown in Scheme W 4A) was purchased from Lakeshore Biomaterials . Baicalin (purity >95%) was purchased from Shanxi Anshun Biotechnology Co., Ltd. . Hgp10025-33 , α-peptide (FAEKFKEAVKDYFAKFWD) and M2-peptide (YEQDPWGVKWWY) were synthesized by GL Biochem Ltd. . CpG  were synthesized by Sangon Biotech . Methylene chloride (AR grade) was purchased from Beijing Chemical Reagent Company . Sodium cholate hydrate from ox or sheep bile (degree of purity ≥ 99%) was supplied by Sigma-Aldrich . Methanol (GR grade), acetonitrile (GR grade), and phosphoric acid (AR grade) were purchased from Beijing Science Experiment Instrument Co., Ltd. . Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco's modified Eagle's medium (DMEM), and fetal bovine serum (FBS) were supplied by Gibco . Fluorochrome-labeled α-CD163, α-CD206, α-MHC II, α-CD86, α-F4/80, α-CD4, α-CD8a, α-CD3e, and α-NK1.1 anti-body for flow cytometry and mouse cytokine ELISA kits were purchased from eBioscience . Antibodies for immunofluorescence staining were purchased from Abcam .Poly   were used as internal aqueous phase. Next, 5 mL methylene chloride containing 100 mg PLGA was employed as oil phase. The primary water in oil (W/O) emulsions were formed by adding the internal water phase to the oil phase under sonication  in a tube held within an ice bath for 40 s at the setting duty-cycle 50%, . The primary emulsions were then added to 30 mL of the external aqueous phase containing 2% (w/v) sodium cholate to form a double emulsion suspension (W/O/W) after a second sonication in a tube held inside an ice bath for 2 min . Finally, the double emulsions were added to 45 mL of aqueous phase containing 0.5% w/v sodium cholate, which were further solidified into nanoparticles (H@NPs and B/H@NPs) by evaporating the dichloromethane under 450 rpm for 4 h at room temperature.PLGA nanoparticles were assembled using an ultrasonic double-emulsion technique -3 M, pH 6.8) with CpG (0.1% w/v) and rotated for 1 h at room temperature to prepare the nano-complexes of B/H@NPs@CpG. Finally, 50 µg M2pep (mp) and 50 µg α-peptide (α) were added to the nano-complexes suspension and rotated for 2 h at room temperature to obtain the nano-complexes that target M2-like TAMs (B/H@NPs@CpG-αmp). The composition details of each group of nanoparticles is shown in PLGA nanoparticle surfaces were coated with polydopamine (pD) (PLGA-pD) to load CpG-ODN and targeting peptides. First, 5 mg baicalin/Hgp nanoparticles (B/H@NPs) were pre-incubated in 1 mL Tris-HCl buffer  containing 2 mg dopamine hydrochloride . The mixture was then incubated at room temperature for 5 h with rotation. Afterwards, the PLGA-pD NPs were collected by centrifugation , and suspended in fresh dopamine (Sigma-Aldrich) solution. They were then incubated with rotation for an additional 3 h at room temperature. The collected PLGA-pD NPs were next incubated in PBS . The drug-loading efficiency (LE) and encapsulation efficiency (EE) of baicalin and Hgp in nanoparticles were calculated as follows:The amounts of CpG loaded on the nanoparticles were analyzed using an Infinite 200 PRO NanoQuant plate .The average size and polydispersity index (PDI) of nanoparticles were measured by a Nano-ZS Zeta Sizer  after resuspending them in deionized water. The morphology of the nanoparticles was characterized using scanning electron microscopy . The structures of B/H@NPs@CpG and B/H@NPs@CpG-αmp complexes were characterized via transmission electron microscopy  at an accelerating voltage of 200 keV. The B/H@NPs@CpG-αmp stability in PBS was monitored by dynamic light scattering (DLS). The release of the drug from the nano-complexes for the case of B/H@NPs@CpG-αmp in PBS (pH 6.5 and pH 7.4) after 7 days was measured by HPLC and a microplate reader.in vitro experiments of cellular function.BMDMs were cultured from bone-marrow cells according to an established protocol 4 cells in 1 mL of culture medium. Second, Nile Red- labelled nano-complexes were added and co-cultured with macrophages for 12 h. Finally, the nano-complexes were taken up into macrophages and observed by CLSM (Leica TCS SPS). The group receiving αmp-blocking was pretreated by administrating saturating concentrations of M2pep and α-peptide. The treated macrophages were fixed in 100 μL 4% paraformaldehyde for 15 min. The cyto-membranes and cell nuclei were then labeled with Alexa Fluor 488-conjugated Phalloidin and DAPI for 20 min, respectively. The samples were observed and imaged by CLSM after washing twice with PBS to remove unbound dye.For the cellular uptake study, macrophages were incubated with the nano-complexes of B/H@NPs@CpG and B/H@NPs@CpG-αmp. First, macrophages were seeded in 20 mm glass-bottom dishes at a density of 5 × 10Flow cytometry (Beckman Coulter CyAn ADP) was used to further quantify the cellular uptake of the B/H@NPs@CpG and B/H@NPs@CpG-αmp nano-complexes. First, the macrophages were seeded in 24-well plates for 12 h, allowing cell attachment to occur. After cells reached 70-80% confluence, they were incubated with Nile Red-loaded nanoparticles for 12 h. The group receiving M2pep-blocking was pretreated by high concentrations of M2pep and α-peptides. Finally, the treated macrophages were washed with PBS to remove excess nano-complexes for analysis by flow cytometry and the mean fluorescence intensity of each cell was measured.4 cells in 96-well plates containing 200 µL growth medium per well. Subsequently, the macrophages were co-cultured with 100 µg aliquots of various nano-complexes for 24 h. Ten µL CCK-8 reagent was then added to each well and further incubated with cells at 37°C for 4 h. Absorbance at 450 nm was measured and the viability results were calculated from the ratio between the average OD450 values of wells containing NP-stimulated cells to the counterparts containing unstimulated cells with medium.Cell viability was assessed using a colorimetric Cell Counting Kit-8 (CCK-8) assay , Cells were plated at a concentration of 2 × 102. The melanoma cells were then seeded in 24-well plates and treated with various nano-complex formulations for 24 h, identified as PBS, Hgp, H@NPs, B/H@NPs, B/H@NPs@CpG, and B/H@NPs@CpG-αmp. Cells were collected after trypsin digestion and centrifuged at 500 × g for 5 min. Ten µL Annexin V-FITC conjugate and 10 µL propidium iodide (PI) were added to each tube and the cells were incubated for an additional 15 min at room temperature in the dark. Finally, the viability and apoptosis levels of cells were determined by flow cytometry.B16 cells were cultured in DMEM supplemented with 10% FBS at 37°C in a humidified atmosphere containing 5% COVarious macrophage phases, such as M0, M1, and M2 types, were induced in RPMI medium 1640 supplemented with different cytokines as follows: M-CSF (20 ng/mL) to induce M0 macrophages; IFN-γ (20 ng/mL) and LPS (100 ng/mL) for induction of M1 macrophages; and IL-4 (20 ng/mL) for induction of M2 macrophages. The various macrophage phenotypes  were then seeded in 12-well Trans well plates (upper compartment) and cultured for 12 h in the same medium. B16 cells were seeded in the lower compartment of each Trans well plate for 12 h. Then, nano-complexes targeting M2-like macrophages (B/H@NPs@CpG-αmp) were incubated with different phenotypic macrophages for 12 h. Finally, T cells were added to the upper compartment of the Trans well plate for 24 h to mimic immune cells with macrophages in the microenvironment. B16 cells in the lower compartment were then collected after trypsin digestion and centrifuged at 500 × g for 5 min. Ten µL Annexin V-FITC and 10 µL PI were subsequently added to each tube for fluorescence labeling. The cells were further incubated for an additional 15 min at room temperature in the dark. Finally, the melanoma cells were centrifuged and washed as preparation for determining cell viability and apoptosis by flow cytometry.All macrophages were detached and removed by scrapping, washed in pH 6.5 PBS, and then counted. After spinning at 1500 rpm for 5 min, 4 million cells were re-suspended in 1 mL baicalin solution  for 10 min. Cells were then separated from the baicalin solution by centrifugation and re-suspended in 7 mL DMEM containing 10% FBS. Tubes containing the cells were placed in a swing bucket at 37°C and centrifuged at 100 rpm. Baicalin incubated cells were extracted after 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h and 6 h intervals of incubation. Drug concentrations remaining in the media were determined by HPLC. Media was completely replaced by fresh media between each sample extraction. Baicalin solution concentrations before and after incubation with cells were also measured to determine the total amounts of drug loading and unloading in each time interval.in vivo experiments. Melanoma implants (B16) were injected subcutaneously in the right armpit of mice and raised for two weeks. The melanoma tumor-bearing mice were given 500 μg Nile Red-loaded nanoparticles (B/H@NPs@CpG and B/H@NPs@CpG-αmp) via intravenous injection (iv.). Tumor tissues were then isolated from tumor-bearing mice at 6 h, 12 h, and 24 h after grafting. For immunofluorescence analysis, tumor tissues were fixed in 4% paraformaldehyde for 12 h at 4°C and then dehydrated in 30% sucrose solution. The tissues were then frozen in Tissue Tek-OTC®  compound and sectioned in 8 µm slices using a freezing microtome . The sections were immuno-stained with FITC anti-mouse CD206  for identification of M2-like TAMs. At the end of processing, the sections were used to measure the co-localization of nanoparticles and M2-like TAMs by means of an automatic multispectral imaging system (PerkinElmer Vectra II). Other harvested tumor tissue extracts were ground into cell suspensions for further analysis by flow cytometry.C57BL/6 mice (6-8 weeks old) were purchased from Vital River Laboratories  and used for 3 (at day 8). The mice were randomly divided in six groups  and intravenously injected with PBS, Hgp, H@NPs, B/H@NPs, B/H@NPs@CpG, and B/H@NPs@CpG-αmp complexes for seven contiguous days , baicalin (3.9 μg), and Hgp (4.8 μg)). Relative tumor volumes (V) were measured every other day. All tumors were excised on day 18 after implantation, and the tumor inhibition rate (R) was calculated as follows:To investigate the anti-tumor efficacy of different formulations, tumor-bearing mice were treated with nano-complexes when the average tumor volume grew to approximately 100 mm5 B16 melanoma cells. Relative tumor volumes (V) were measured every two days. All tumors were excised on day 18 after implantation.To investigate the immunization efficacy of the nano-complexes against melanoma, the above nano-complexes were used to trigger macrophage activation in normal C57 mice on a continuous daily schedule for one week , baicalin (3.9 μg), and Hgp (4.8 μg)). One day after the last vaccination, the mice were inoculated subcutaneously with 5 × 10Cytokine levels in serum and at tumor sites were quantified using mouse α-IL-10, α-IL-12p70, α-IFN-γ, α-IL-6, α-IL-2 and α-TNF-α ELISA kits following the manufacturer's protocol (eBioscience). Raw data were calculated using a five-parameter curve obtained from the absorbance values of standards provided by the manufacturer.Tumor cell suspensions from six mice were prepared, and TAMs markers  were identified by flow cytometric analysis for evaluation of TAMs phase reversion. In addition, T-lymphocyte markers (CD4 and CD8) and NK cell marker (NK1.1) were used to identify T-lymphocyte and NK cell infiltration in the tumor microenvironment. Tumor tissues from three animals of each group were frozen for immunofluorescence staining. Immunofluorescence analysis was used to evaluate TAMs reversion, together with T-lymphocyte and NK cells infiltration. The frozen sections were immuno-stained with FITC anti-mouse CD206 (Abcam) and with Rhodamine-labeled anti-mouse CD86 for TAM reversion. FITC tagged anti-mouse IFN-γ, anti-mouse CD8, and anti-mouse NK, were used to identify immune cells infiltration measurements. In addition, the frozen sections were antibody stained using anti-mouse casepase-3, α-CD31, α-VEGF, and α-MMP9 for assessing tumor cell apoptosis and metastasis. After processing, all stained sections were measured by the automatic multispectral imaging system (PerkinElmer Vectra II). The immunofluorescence analysis was used to evaluate whether the tumor microenvironment was remodeled by M2-like TAMs reversion and infiltration of T and NK cells.th day after tumor seeding, tissues were collected from the transplant and analyzed using hematoxylin and eosin (HE) staining. Hearts, livers, spleens, lungs, and kidneys were harvested from tumor-bearing mice and fixed in 4% paraformaldehyde solution. The organs were embedded in paraffin, sectioned, and processed for HE staining. The sections were measured by an automatic multispectral imaging system (PerkinElmer Vectra II).On the 18All animal experiments were performed in compliance with the guide for care and use of laboratory animals. The animal protocol was approved by the Institutional Animal Care and Use Committees at Institute of Process Engineering, Chinese Academy of Sciences.t-test. Differences among multiple groups were tested with one-way ANOVA followed by Tukey's multiple comparison. Significant differences between groups are expressed as follows: *P < 0.05, **P < 0.01, or ***P < 0.001.All results are expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed using GraphPad Prism 5.0 software . Differences between two groups were tested using an unpaired, two-tailed, Student's The nano-complexes containing baicalin and/or Hgp antigen (H@NPs and B/H@NPs) were fabricated using a double-emulsion technique combined with solvent evaporation. In Figure The amounts of baicalin and Hgp encapsulated within the formulations were determined using high-performance liquid chromatography (HPLC). The encapsulation efficiency for baicalin and Hgp was 44.3 wt% and 30.2 wt%, respectively A, B. TheThe structures of the nano-complexes including B/H@NPs and B/H@NPs@CpG-αmp were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). A spherical core-shell structure was observed in the image of B/H@NPs Figure B as expeThe B/H@NPs@CpG-αmp nano-complexes were suspended in phosphate-buffered saline  to evaluate their stability. The particle size was continuously monitored via DLS for 7 days. The nanoparticles surface zeta was measured by a Nano-ZS Zeta Sizer also for 7 days under the same conditions. The zeta potential (green line) of B/H@NPs@CpG-αmp increased from -17.8 ± 0.3 mV to -16.2 ± 0.4 mV, and the size (orange line) slightly increased from 117.6 ± 0.6 nm to 128.8 ± 0.7 nm, as shown in Figure in vitro release of baicalin, Hgp, and CpG from the nano-complexes was investigated by resuspending nano-complexes in PBS at pH 6.5 (close to the pH of the tumor microenvironment). There were two stages in the release curves. As seen in Figure in vitro release of baicalin, Hgp, and CpG from nano-complexes was investigated in PBS at pH 7.4 . Our results, as shown in Figure in vivo. The above results demonstrate that the nano-complexes designed in this study can be effectively loaded with baicalin, antigen, and CpG; exhibit good storage stability in PBS; and undergo slow release of payload in an acidic environment. The data suggest that the nano-complexes should achieve extended-release of payloads in a weak acid, tumor environment. These properties give support to our belief that these formulations will be found effective in countering the phenotype reversal in macrophage differentiation and thereby lead to prolonged anti-tumor responses.The In order to remodel the tumor microenvironment by reprogramming M2-like TAMs using nano-complexes, it was crucial to transport the payload to M2-like TAMs as a first step before its internalization. Recently, M2pep (mp) and α-peptide (α) were reported to be specific ligands for targeting M2-like TAMs in vitro competition experiments to test the uptake of B/H@NPs@CpG-αmp nano-complexes by M1-like macrophages and M2-like macrophages. For the competition assay, M1-like and M2-like macrophages were seeded in 12-well plates in a 1:1 ratio. Then, B/H@NPs@CpG-αmp labeled with Cy5 was co-incubated with the mixed macrophages for 12 h. Next, the amounts of nano-complexes taken up by M1-like and M2-like were detected by flow cytometry. As shown in in vitro.Accordingly, we selected the nano-complexes (B/H@NPs@CpG-αmp) containing dual targeting peptides for evaluation of M2-like macrophage-targeting. Confocal imaging data showed that B/H@NPs@CpG-αmp displayed remarkably stronger fluorescent intensity in M2-like macrophages than the B/H@NPs@CpG complexes that lack of targeting ligands . As shown in Encouraged by the excellent uptake of nano-complexes in M2-like macrophages and the validation of the low toxicity, we next investigated the capacity of nano-complexes for killing B16 cells. It has been reported that baicalin inhibits Hela cells, lymphocytic leukemia, lung cancer, ovarian cancer, and human colorectal cancer cells in vitro using Trans well plates with tumor cells, TAMs and T cells. As shown in the summarized results of Figure vs M1 vs M2 vs phenotype mix) treated with nanoparticles without baicalin and CpG and evaluated tumor cell apoptosis. The results are shown in Figure Some studies report that CpG carries out an anti-tumor role through activating immune cells and reversing M2-like macrophage phenotypes in a tumor microenvironment (M2-like TAMs). The activated TAMs subsequently secrete additional cytokines and present antigen to T cells that induces the anti-tumor immune response In the above experiments, baicalin and CpG were important for the anti-tumor response. We next identified the phenotypes of macrophages that were incubated with the nano-complexes. As shown in Figure in vitro and in vivo. The mixed macrophage group produced the highest tumor cell apoptosis among all the groups  can be delivered to tumors using macrophages as carriers. Chemotherapy-loaded macrophages showed promising anti-cancer efficacy in terms of tumor suppression, life span prolongation, and metastasis inhibition, with reduced toxicity s Figure B. This iin vitro. We next evaluated the targeting capability of B/H@NPs@CpG-αmp in vivo. To localize the nano-complexes, B/H@NPs@CpG and B/H@NPs@CpG-αmp, in vivo, they were labeled with fluorescent Nile-Red dye, and their localization at the tumor site was tracked. As expected, cryosections of tumor tissues showed that 6 h to 24 h after injection, B/H@NPs@CpG-αmp (red) nano-complexes co-localized with CD206+ TAMs (green)  cells into the right armpit of mice. After two weeks, the mice were intravenously given 100 μL Cy5-labeled formulations including Free CpG, B/H@NPs@CpG (non-targeting), B/H@NPs@CpG-mp (single peptide-targeting) and B/H@NPs@CpG-αmp . Then, the dynamic distributions of different formulations in various organs were monitored using an in vivo imaging system  and flow cytometry. As shown in We also tracked thein vivo. These observations pave the way for achieving specific delivery of therapeutic drugs  to M2-like TAMs in vivo.Before 24 h, most of the non-targeting formulations, such as the B/H@NPs@CpG nano-complex mainly distributes in liver, spleen and lungs, with only a small proportion reaching the tumor site, presumably due to the EPR effect. However, both single-targeted and double-targeted nano-complexes, such as B/H@NPs@CpG-mp and B/H@NPs@CpG-αmp, reach the tumor sites quickly, and further accumulate there gradually over time. This pattern is especially evident for the dual-targeted nano-complexes, such as B/H@NPs@CpG-αmp. After 72 h, a large amount of the dual-targeted nano-complexes accumulated at the tumor site, and little remained in liver . The dynamic distribution of the dual-targeting nano-complexes to various organs was also confirmed by flow cytometry data. These measurements show that the nano-complex accumulation at the tumor sites increased gradually over time C. Taken  in vitro and target M2-like TAMs in vivo. Here, the polarization and activation of TAMs in melanoma-bearing mice was examined using flow cytometry and immunofluorescence staining. The transformation of TAM surface co-stimulatory molecules reflects the phenotype reversion. For example, M1-like TAMs are associated with MHC II and CD86 surface markers, and M2-like TAMs with CD163 and CD206 markers. As shown in Figure The preceding results show that B/H@NPs@CpG-αmp nano-complexes effectively convert M2-like phenotypes into M1-like phenotypes+ cells with the hatch-filled grid, and CD206+ cells with the solid filled color). These results indicate that the ratio of M1-like to M2-like TAM phenotypes within tumor tissue is conspicuously enhanced after exposure to the nano-complexes . This result suggests that baicalin and CpG-loaded nanoparticles also play a role in reversing M2-like to M1-like conversion of TAMs. It is surprising that the B/H@NPs@CpG-αmp nano-complexes promote greater phenotype conversion from M2-like to M1-like compared with B/H@NPs@CpG (from 1:0.4 to 1:0.87). Initially, the nano-complexes with dual peptide targets, such as B/H@NPs@CpG-αmp, are engulfed predominantly inside M2-like TAMs. Subsequently, the B/H@NPs@CpG-αmp releases most of its CpG into the target cells, which then carry out M2-like to M1-like reversion.Figure omplexes C. B/H@NPBased on TAM phenotype reversion, cytokine expression levels are also important for evaluating the tumor microenvironment The important cytokines for TAMs polarization and T cell activation were IL-2 and IFN-γ + T cells 6.02-fold and CD8+ T cells 1.3-fold over the PBS group), as shown in Figure + T cells) and CTL (CD8+ T cells) lymphocytes within tumor tissues are significantly elevated by B/H@NPs@CpG and B/H@NPs@CpG-αmp. The above results suggest that the cellular events occur in the following order: TAMs phenotype reversion is first induced by CpG and baicalin. M1-like TAMs overcome the barrier of antigen presentation and further recruit and activate additional T cells. Nano-complexes of B/H@NPs@CpG-αmp targeting M2-like TAMs showed the greatest percentage of both CD4+ T cells and CD8+ T cells in the tumor microenvironment. The CD4+ T cell percentages increased from 6.02-fold to 13.3-fold  inhibited the proliferation of hepatoma H22 cells through induction of cytochrome C, activation of caspase-3, procaspase 9, and the FNFR superfamily. In the present study, we determined the expression of caspase-3 in tumor tissues by immunofluorescence staining. The immunofluorescence images of Figure Figure The antitumor effect and immunomodulatory response of free baicalin, free CpG, and free baicalin and CpG (free B&C) were investigated in this study. The results  do not sin vivo, respectively . The nano-complexes and other formulations were injected 7 times at one day intervals starting 8 days after B16F10 cell implantation  A. The tectively -E. Theseectively C-D. HoweB16 melanoma is highly aggressive and straightforwardly metastatic. The capacity of nano-complexes to inhibit tumor metastasis was investigated. Metastatic melanoma cells were found in spleen in varying degrees for the PBS, Hgp, and H@NP treated animals Figure A. We useet al. reported that M2-like TAMs reversion greatly decreased VEGF and MMP9 expression in tumor tissues Therefore, tumor microenvironment remodeling also inhibits metastases by the dual mode model (M2-like TAMs phenotype reversion and immune cells activation), which was shown to be active for the nano-complexes of B/H@NPs@CpG-αmp. These modes were found to be favorable for promoting pro-inflammatory cytokines secretion and anti-tumor immunity. Huang in vivo, we investigated their effects as a preventative formulation. Six different nano-complexes were used to immunize normal C57 mice for 7 consecutive days  was evaluated by immune histochemical analysis. As shown in The in vitro and in vivo studies showed that the as-prepared nano-complexes successfully targeted and localized at M2-like TAMs and reversed their phenotypes to M1-like phenotypes. The activated M1-like TAMs acted as antigen presenting cells to present Hgp antigens to neighboring T cells for further activation of the inflammatory cascade to enhance the tumor killing function.A nano-complex that targets M2-like macrophages was successfully formulated for melanoma treatment. The nano-complex was shown to effectively reverse the TAM phenotype phase and further remodel the solid tumor microenvironment. PLGA nanoparticles were fabricated to encapsulate baicalin and/or Hgp antigen, and their surfaces were coated with polydopamine for ligand attachment. The nanoparticles were further decorated with M2pep and α-peptide ligands to form nano-complexes that target M2-like TAMs. The results of the The reversion of M2-like TAMs to M1-like phenotype and activation of T cells significantly remodeled the tumor microenvironment in such manner that it resulted in immune cell recruitment and enhanced anti-tumor therapy. The substantial retardation of tumor growth was observed in tumor-bearing mice after intravenous administration of the nano-complexes for one week. Collectively, these results suggest that the TAM-targeting nano-complexes offer significant potential for treating and preventing melanoma by reversing M2-like TAMs and remodeling the tumor microenvironment.Supplementary figures and table.Click here for additional data file.


Caenorhabditis elegans, CUC-1, in distal tip cell migration, which is a key process during the development of the C. elegans gonad. Using knock-out worms, in which the cuc-1 gene was removed by CRISPR-Cas9 technology, we probed life span, brood size, as well as distal tip cell migration in the absence or presence of supplemented copper. Upon scoring of gonads, we found that cuc-1 knock-out, but not wild-type, worms exhibited distal tip cell migration defects in approximately 10–15% of animals and, had a significantly reduced brood size. Importantly, the distal tip cell migration defect was rescued by a wild-type cuc-1 transgene provided to cuc-1 knock-out worms. The results obtained here for C. elegans CUC-1 imply that Atox1 homologs, in addition to their well-known cytoplasmic copper transport, may contribute to developmental cell migration processes.Cell migration is a fundamental biological process involved in for example embryonic development, immune system and wound healing. Cell migration is also a key step in cancer metastasis and the human copper chaperone Atox1 was recently found to facilitate this process in breast cancer cells. To explore the role of the copper chaperone in other cell migration processes, we here investigated the putative involvement of an Atox1 homolog in  At the larval stage L2, the DTCs migrate away from the gonad primordium. At L3, the DTCs turn and migrate to the dorsal side, followed by a U-turn and migration towards the mid-body in the L4 stage  C. elegans is transparent. Moreover, the C. elegans genome is conserved, which allows translation of functions to other systems, including humans. Dysfunctional DTC migration may result in a range of gonad development defects, such as turning, pathfinding and cessation problems which are easily detected under a microscope  that can be rescued by wild-type cuc-1 provided as a transgene. Our results suggest that CUC-1 is a co-regulator of DTC migration during C. elegans larvae development.In this study, taking advantage of the DTC migration Caenorhabditis elegans was cultivated at 20 °C on nematode growth medium (NGM) plates with E. coli OP50 (worm base ID WBStrain00041969) as the food source. E. coli OP50 is an uracil auxotroph whose growth is limited on NGM plates. A limited bacterial lawn is desirable because it allows for easier observation and better mating of the worms. The media, culture and handling of C. elegans follow what was described by Brenner , and the PHX1006(cuc-1(syb1006)/hT2)) strain was created by Suny Biotech  using CRISPR/Cas9 to delete cuc-1  was measured using the ImageJ software  and Synchronous L1 worms were plated onto NGM plates with OP50 and after an additional 48 h of growth (to reach L4 stage), 10 worms were singled out onto new plates. The worms were transferred daily during the fertile period and live progeny were counted 3 days after removal of the hermaphrodite.cuc-1 mutant strains. The worms were transferred every second day during the fertile period and once a week thereafter. All worms were monitored every day and scored as dead when failing to respond upon several touches on the head with the worm-pick.Synchronous L4 worms were plated in groups of five onto NGM plates with OP50. 100 worms were picked for both N2 and Synchronous L1 worms were plated onto NGM plates with OP50, and after an additional 72 h when grown to young adults, hermaphrodites were observed on 2% agar pads using a Zeiss Axio photo microscope equipped with a 60X objective and Nomarski optics (Sulston and Horvitz pCUC-1 rescue construct was generated in-house with a Gibson assembly kit (Gibson Assembly® Cloning Kit (New England Biolabs)) using the following primers.The cuc-1 gene including 2 kb of upstream regulatory sequence using N2 worm genomic DNA as template.5′- GGAATTCGCCCTTGTCTAGAGTAAGCTATTTTAGAGATTTTTGAC-3′ and 5′- TATCTGCAGAATTCGCCCTTACGTTTACCTTTCTGAGAAG − 3′ to amplify the pPAQR-2::GFP construct /hT2) worms at 10 ng/µl together with 5 ng/µl pPD118.33 (Pmyo-2::GFP) (Addgene plasmid #1596) and 85 ng/µl pBSKS (Stratagene).The plasmid construct was confirmed by sequencing using 5′-GTGGCGGCCGCTCTAGAACTAGTAACGGCCGCCAGT-3′ and 5′-TAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGA-3′. The assembled t-tests were used to determine statistical significance of our measures. All experiments were repeated at least three times. Asterisks presented in the figures indicate various degrees of significance, as follows: *p < 0.05, **p < 0.01, ***p < 0.001.The Student’s C. elegans worms with the cuc-1 gene deleted (cuc-1 mutant) using CRISPR-Cas9 technology  than wild-type worms  were classified as pathfinding defects.Next, we analyzed the DTC migration patterns by scoring wild-type and cts Fig.  d,e, whects Fig. b observeE. coli OP50 food) does not include added Cu, although the E. coli and the yeast extract in the agar are sources of Cu and thus the worms have access to some Cu (but exact concentrations are not known). Previous studies have shown that removal of all Cu by addition of chelation compounds, or addition of excessive levels of Cu to the growth media, diminish growth of C. elegans, but for up to 100 µM of Cu supplemented in the NGM agar, there are no growth defects noted in worms  which, due to regulation of cellular Cu uptake and transport in worms  into cuc-1 mutant worms. First, pCUC-1 was injected into the gonad syncytium of cuc-1 mutant worms to establish a transgenic line. Transgenic worms in subsequent generations were verified by PCR , function in Cu transport as in humans, but with tissue-specific expression , we found pathfinding problems to be most common followed by turning problems. However, our classification gives only rough estimates as, in some cases, more than one problem may be present, and the total number of defective worms analyzed are small. The results for CUC-1 deletion should be placed in relation to similar studies investigating the roles of other gene products in DTC migration. Reporting migration defects as a percentage of worms with defects is a common way to quantify dysfunction due to gene mutations. In other studies, the percentage of worms with DTC migration defects range from around 10–20% defective worms to much higher percentages, depending on the gene deleted. Rho GTPases such as CED-10 and MIG-2 are known to play key roles in DTC migration  in 27% and 12% of worms, respectively  for CUC-1.We found that whereas wild-type worms had no DTC migration defects at the two conditions studied, approximately 10%  and 15% (50 µM Cu supplemented to the plates) of C. elegans did not affect brood size, worm length, or DTC migration at added Cu concentrations up to 100 µM  in C. elegans development. Thus, CUC-1 should be considered a new modulator of the extensive network of proteins that facilitate DTCs migration in worms. CUC-1 may play Cu-dependent or Cu-independent roles in DTC migration. We speculate that there may be unknown Cu dependencies for other proteins in these processes to which CUC-1 would deliver Cu. Speculatively, Cu-loaded CUC-1 may help mediate oxidation/reduction processes that regulate Rho GTPases implicated in DTC migration, such as Rac, Rho, and Cdc-42. In human cells, Atox1 was found to mediate cancer cell migration by (via Cu transport) promoting extracellular, Cu-dependent, collagen-remodeling lysyl oxidase (LOX) activity. C. elegans worms do not have LOX proteins but instead depend on other proteins that regulate the extracellular matrix. No Cu dependence for those proteins have been reported but, nonetheless, many are oxidases, peroxidases and thioredoxin-like proteins (Myllyharju and Kivirikko Our findings demonstrate that the Atox1 homolog in worms, CUC-1, plays a role in promoting correct DTC migration during


Patients with the progression of the disease show immune dysfunction. We aim at studying whether miR-181b is also involved in this process. We demonstrate that miR-181b can be increased in CLL cells by crosstalk with CD40L+ T cells, enhancing the maturation of CD8+ T cells in cytotoxic T lymphocytes (CTL) and, in turn, the anti-tumor cytotoxic T cell response in in vitro and in vivo models. These results demonstrate a role of the miR-181b in the immune response against tumor and envisage a therapeutic action of miR-181b in a specific milieu surrounding CLL cells.Low expression of miR-181b-5p (miR-181b), promoting the death of CLL cells. Here we investigated whether the reduction of miR-181b impairs the immune response in CLL. We demonstrate that activated CD4+ T cells increase miR-181b expression in CLL through CD40–CD40L signaling, which enhances the maturation and activity of cytotoxic T cells and, consequently, the apoptotic response of CLL cells. The cytotoxic response is facilitated by a depletion of the anti-inflammatory cytokine interleukin 10, targeted by miR-181b. In vivo experiments in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice confirmed that miR-181b promotes the apoptotic death of CLL cells only when functional T cells are restored. Overall, our findings suggest that the reinstatement of miR-181b in CLL cells could be an exploitable adjuvant therapeutic option for the treatment of CLL.The clinical progression of B cell chronic lymphocytic leukemia (CLL) is associated with immune cell dysfunction and a strong decrease of  In Chroignaling ,7. T celignaling .BCR signaling is constitutively activated in CLL cells ; therefomiR-181b, a regulator of B cell differentiation, is expressed at low levels in CLL cells [miR-181b decrease during disease progression in peripheral blood mononuclear cells (PBMCs) from CLL patients [miR-181b reduces the leukemic cell fraction [MiR-130a and miR-181a are also able to induce the death of primary CLL cells in vitro [MicroRNAs (miRNAs) take part in the immunological key processes of innate and adaptive immune cells , and theLL cells , which aLL cells ,17. We pin vitro , and thein vitro .miR-181b, miR-181a, and miR-130a that occur in patients during CLL progression could be due to the impaired interactions between T and CLL cells. To this end, we tested whether, potentiating such interactions, the expression of miR-181b, miR-181a, and miR-130a could be restored and whether restoring the expression of miR-181b could promote cell-mediated immune response and apoptosis in CLL cells.We hypothesized that the low expression of miR-181b, miR-181a, and miR-130a observed in CLL could reflect inefficacy of T cell activation, we co-cultured CLL cells with CD4+T cells from healthy donors, since T cells from CLL patients show pathological defects [To investigate whether the decreased expression of  defects ,21. Rest defects     A–C. In aon test) D–F.+T cells increased miR-181b expression in CLL cells    A,B and aon test) D,E. This-miR-130 C,F.+T cells from healthy donors, activated or not, were co-cultured in a transwell system with patient-derived CLL cells from the above experiment  to block CD40-CD40L interactions , as previously described [miR-181b staining  or control (LV CTRL) were generated and tested  by mixing CD40L-stimulated MEC-1 and healthy allogenic T cells [miR-181b (LV AS miR-181b)   after the CTL generation assay. We noted that in one of the two-studied case, CTLs were less abundant when T cells were co-cultured with miR-181b-depleted MEC-1 cells, and T lymphocytes had lower cytotoxic activity when mixed with fresh third-party MEC-1 cells  . After c control A. To ass chamber B. AccordB (GrzB) . Next, w-1 cells B,D. Varit ratios .miR-181b in CLL cells promotes activation and CTL maturation of T cells via direct B-T interactions.Overall, these findings indicate that enhanced expression of miR-181b, we studied factors that might modulate CTL maturation. We focused on IL10, which is known to have a role in the suppression of cell-mediated immune response [IL10 mRNA expression in purified primary CLL cells overexpressing miR-181b . In 2 of the 13 mice pairs that received human T cells, subsequent inoculation of MEC-1 cells did not lead to detectable GFP+ cells in the spleen or bone marrow; therefore, these mice were excluded from the analysis. Six of the remaining 10 mice pairs that received miR-181b, showed lower GFP+ cells and higher CD3+ cells in spleens and bone marrows compared to the controls  Exogenous miR-181b, by targeting the anti-apoptotic proteins MCL-1 and BCL-2, sensitizes CLL cells to the extrinsic apoptosis through CD8+ T cells; and (ii) miR-181b reduces the expression of IL10, which inhibits the production of cytokines that promote the generation of effector CTLs [CLL cells do not undergo apoptosis, despite the fact that their BCRs are constitutively activated . This cotor CTLs ,34,35 . Stimulation of CLL cells by activated T cells may not always increase miR-181b expression, suggesting the existence of a subset of refractory patients with different cytotoxic T cell response that may be influenced also by the patient genetic trait [miR-181b in CLL cells, concomitant with T cell activation or CD40-CLL stimulation, could be therapeutically effective for the above-described refractory patients and a valid alternative for the management of all CLL patients.In vitro results are strongly supported by in vivo experiments in that enhanced expression of reported ,43. Howereported , they tr T cells   and control non-neoplastic tonsil tissues were provided by Policlinico Agostino Gemelli (Rome). Blood from healthy donors was collected at the SS. Annunziata Hospital (Chieti). The characteristics of the CLL patients studied are reported in 2 incubator and the media were supplemented with 10% fetal bovine serum, 1% Pen/Strep, and 1% L-glutamine .HeLa cells expressing CD40 ligand (CD154) (HeLa-CD40L or HeLa-CD154) were previously described  and kindPeripheral blood mononuclear cells were isolated from whole blood by density gradient centrifugation using Ficoll-Paque PLUS . CD4+, CD8+, or CD3+ T cells were isolated from PBMCs using the CD4+ T Cell, CD8+ T Cell, or Pan T Cell isolation kits . CLL cells were obtained from PBMCs, using the B-CLL Cell Isolation Kit , or from whole blood, using the RosetteSep Human B Cell Enrichment Cocktail . CD34+ cells were enriched from umbilical cord blood using the CD34 Microbead Kit .6 cells/well) and activated for 3 days using the human T Cell Activation/Expansion Kit . Activation of CD4+ T cells was assessed by CD40L expression using flow cytometry (data not shown). Purified CLL cells were co-cultured at 1:1 ratio with CD4+ T cells for 4 and 24 h, then CLL cells were isolated and RNA was analyzed for gene expression. The purity of isolated CLL cells was verified by flow cytometry, which showed an average of 98.9% (co-culture with not-activated T cells) and 98.7% (co-culture with activated T cells). Monoclonal anti-human CD40L antibody was added to the T cell cultures 24 h before T-B cell contact to inhibit interactions; normal mouse IgG was used as the negative control  and cultured for additional 24 or 48 h. Then, CLL cells were isolated and RNA was analyzed for gene expression.PBMCs from CLL patients were seeded in X-VIVO medium  containing 5% human serum  at the final concentration of 3.5 × 10Total RNA was extracted using QIAzol Lysis Reagent , quantified by spectrophotometry on a Nanodrop 2000 instrument  and assessed for quality by the RNA HighSens Analysis Kit in the Experion Automated Electrophoresis System . For pri-miRNA analysis, total RNA was treated with DNase .RNU44, ACTB, or GAPDH as appropriate by the 2−Δct method.Total RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit  and quantitative PCR was performed using the FastStart Universal Probe Mastermix kit  or the QuantiFast SYBR Green PCR kit  for IL10. Fragments were amplified using the CFX96 Touch Real-Time PCR System . Primer sequences and FAM probes were designed using UPL Assay Design Center Roche Software . Gene exIn situ hybridization (ISH) was performed on formalin-fixed, paraffin-embedded, 4 µm-thick serial sections mounted on positively charged slides from lymph nodes of CLL patients and tonsils of non-cancer patients. Histological sections were deparaffinized, hydrated, washed in PBS, incubated with proteinase K , washed again in PBS, dehydrated, and incubated overnight at 42 °C with miRCURY LNA detection probes . After iNuclear extraction from CLL cells was performed using the NE-PER Nuclear and Cytoplasmic Extraction kit . Nuclear fractions were dissolved in QIAzol Lysis Reagent  for RNA extraction and gene expression analyses.5 cells were washed in Washing Buffer , resuspended in 1 mL of FACS Lysing solution 1X , mixed and incubated at room temperature in the dark for 10 min, washed in WB, resuspended in 1 mL of permeabilizing solution 2 1X , and mixed and incubated at room temperature in the dark for 10 min. Then, cells were washed with WB, stained with the GrzB antibody for 30 min at 4 °C in the dark, finally washed in WB, and resuspended in 500 µL of PBS1X.Surface B and T cell markers were assessed by flow cytometry using the antibodies reported in Data were acquired with a FACSCanto II cell analyzer  and analyzed with FlowJo v10.0.7 and/or FCS Express 5 software .miR-181b2, was amplified by PCR from HEK293T cells using specific primers , and either pTween_181b2 or pTween_CTRL using the Calcium Phosphate Transfection Kit . After 48 h, the culture supernatants were harvested, cleared by centrifugation at 1500 rpm at room temperature for 10 min, and filtered through a 0.45 µm syringe filter. The obtained lentiviruses were named LV miR-181b and LV CTRL.To generate the lentivirus expressing Lentiviruses expressing miR-181b antisense (AS) sequences, or the control, were generated using pAS miR-181b or pCTRL and the pPACKH1 HIV Lentivector Packaging Kit . The obtained lentiviruses were named LV AS miR-181b and LV AS CTRL. Virus titers were assessed by the percentage of GFP-positive cells using flow cytometry, according to the formula: Viral particles/µL = (no. of cells) × (% GFP-positive cells)/volume of virus solution.MEC-1 cells were infected with lentiviruses at 10 multiplicity of infection (MOI), while CLL cells or PBMCs were infected at MOI 0.5. Briefly, cells were suspended in medium containing lentivirus and polybrene (10 μg/mL), seeded in 6-well plates, and centrifuged for 45 min at 1800 rpm. After 24 h, the virus-containing medium was replaced with a complete culture medium. Transduction efficiency was determined from the percentage of GFP-positive cells using flow cytometry or by measuring the transducer gene miR-181b using RT-qPCR.5/well and co-cultured with purified CD3+ T cells from healthy donors at an effector/target (E/T) ratio of 2:1 in serum-free AIM-V. After 4 days of B and T lymphocytes co-culture, cells were supplemented with IL-2  and cultured for additional 4 days. On day 8, T cells were harvested and analyzed by flow cytometry, used as effector cells in CTL assays, or mixed with CLL cells for the second round of CTL generation.Effector cytotoxic lymphocytes (CTLs) were generated as described by Chu et al. , with so4) with either LV miR-181b or LV CTRL at the E/T ratios 1:1, 5:1, 10:1. After 4 or 7 h, MEC-1 cells were labeled with Annexin-V and analyzed by flow cytometry  pre-labeled with Violet Proliferation Dye 450  in AIM-V medium at different E/T ratios for 1 or 2 h at 37° C. After co-culture, isolated MEC-1 cells were stained with Annexin V and analyzed by flow cytometry  with or without activated T cells from healthy donor 22  in RPMI medium supplemented with 20% FBS. MEC-1 cells infected with either LV AS miR181b or LV AS CTRL were stimulated on HeLa-CD40L for 3 days and co-cultured into the upper chamber of transwell dishes with or without activated T cells from healthy donor 22 , as in the generation of effector CTLs. Activated and non-activated T cells in the upper and lower chambers, respectively, were from the same healthy donor. T cells in the lower chamber were cultured at the same E/T ratio as in the upper chamber. After 8 days of incubation, the T cells in the lower chamber were analyzed by flow cytometry.MEC-1 cells infected with LV miR-181b or LV CTRL were seeded at 1.8 × 10miR-181b, or MEC-1 silencing miR-181b, and their relative controls, were activated on HeLa-CD40L cells for 3 days, then cells were cultured at different conditions  and the supernatant was assayed for the presence of IL10.IL10 was measured in the culture medium using the Human IL-10 ELISA Ready-SET-Go kit . Primary CLL cells overexpressing 6 cells), miRNA mimics (50 pmol for 1 x106 cells) or siRNA (50 pmol for 1 × 106 cells) in MEC-1 cells were performed using the U-016 program of the Amaxa Nucleofector II . Transient transfections of plasmids (2µg for 3–5 × 106 cells) in primary CLL cells were performed using the U-016 program of the Amaxa Nucleofector II .Transient transfections of plasmids (2 µg for 1 × 10IL10 promoter activity was measured in MEC-1 cells. FOS expression vector pLX304-FOS-V5 (pFOS)   or contrCells were collected and lysed in cold protein lysis buffer  supplemented with Protease and Phosphatase Inhibitors Cocktail 3 . Cells lysates were run on Criterion TGX Stain-Free pre-cast gels  and blotted on Immun-Blot PVDF membranes . Primary and secondary antibodies are reported in 7 MEC-1 cells infected with either LV miR-181b or LV CTRL; 10 sibling pairs were injected intravenously with 106 healthy T cells and, after 1 week, with infected MEC-1 cells; one sibling pair was injected with 105 enriched CD34+ cells from human cord blood and, after 6 months, with infected MEC-1 cells. In both experiments, mice were euthanized by CO2 inhalation 4 weeks after MEC-1 cells injection. Spleens were homogenized and filtered through 40-µm cell strainers. Bone marrow cells were obtained by flushing the femora, tibiae, and hip bones with PBS and then filtering through 40-µm cell strainers.NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice  of both sexes were bred and housed under specific pathogen-free conditions in temperature-controlled cages at the institutional animal facility of the G. d’Annunzio University. Animal experiments followed the regulations of the Italian law and the project, which predated the adoption of the d.lgs. n. 26/2014, was approved by the Interinstitutional Committee on Animal Research and Ethics of the Universities of Chieti-Pescara, Teramo, L’Aquila and the Experimental Zooprophylactic Institute of Abruzzo-Molise (CEISA) (Protocol UNCHD12#222/2014). Four/eight-week-old mice were used. Six sibling pairs of mice were injected intravenously with 10Spleen samples were fixed in 10% buffered formalin and embedded in paraffin; 5 µm sections were stained with hematoxylin and eosin . After antigen retrieval at pH 9 for 20 min in a microwave, blank serial sections were incubated with the antibodies reported in t-test or Mann–Whitney U-test and paired t-test or Wilcoxon matched-pairs test were used. Pearson’ coefficient was used to analyze the linear statistical dependency of two variables. Statistical tests were two-sided; p-values ≤ 0.05 were considered significant.Statistical analyses were performed using GraphPad Prism 6.01 . Normality of distributions data was assessed by the D’Agostino & Pearson test; based on the normality test, unpaired https://github.com/VeroneseVisoneLabs).All the raw data are freely available at the code-hosting platform GitHub (miR-181b in CLL cells is involved in the immune dysfunction that characterizes this disease. Restoration of the physiological miR-181b activity in CLL cells may be a challenging novel approach to treat CLL patients.We demonstrate that the down-regulation of


Background: Hepatic myofibroblasts (MFs) can originate from hepatic stellate cells, portal fibroblasts, or bone marrow-derived mesenchymal stem cells and can migrate towards the site of injury by aligning with nascent and established fibrotic septa in response to several mediators. Oncostatin M (OSM) is known to orchestrate hypoxia-modulated hepatic processes involving the hypoxia-inducible factor 1 (HIF-1). Methods. In vivo and in vitro experiments were performed to analyze the expression of OSM and OSM-receptor (OSMR) in three murine models of non-alcoholic-fatty liver disease (NAFLD) and -steatohepatitis (NASH) and in human NASH patients as well as the action of OSM on phenotypic responses of human MFs. Results: Hepatic OSM and OSMR levels were overexpressed in three murine NASH models and in NASH patients. OSM stimulates migration in human MFs by involving early intracellular ROS generation and activation of Ras/Erk, JNK1/2, PI3K/Akt as well as STAT1/STAT3 pathways and HIF-1α. OSM-dependent migration relies on a biphasic mechanism requiring early intracellular generation of reactive oxygen species (ROS) and late HIF1-dependent expression and release of VEGF. Conclusion: OSM is overexpressed in experimental and human progressive NAFLD and can act as a profibrogenic factor by directly stimulating migration of hepatic MFs. During chronic liver diseases (CLDs), liver fibrogenesis is sustained by an heterogeneous population of myofibroblast-like cells (MFs) that can originate mainly from hepatic stellate cells (HSC/MFs) and portal (myo)fibroblasts or, to a less extent, circulating and bone marrow-derived mesenchymal stem cell (MSCs) engrafting chronically injured liver ,3,4,5,6.Materials: The methionine and choline-deficient (MCD) diet, the choline-deficient amino acid-refined (CDAA) diet, the high fat–high fructose diet (HFHF) as well as the related control diets, methionine choline supplemented (MCS) diet and choline-supplemented and amino acid-refined (CSAA) diet and normal (not supplemented with fat) diet were provided by Laboratorio Dottori Piccioni srl . The kit for RNA retro-trascription and for real time PCR were purchased from Biorad . Enhanced chemiluminescence (ECL) reagents, nitrocellulose membranes (Hybond-C extra) were from Amersham Pharmacia Biotech Inc. . Human recombinant Oncostatin M (OSM) was from Petrotech . Monoclonal antibody against p-ERK1/2 (sc-7383), polyclonal antibody for p-STAT1 (sc-8394), STAT1 (sc-346), p-STAT3 (sc-8059), STAT3 (sc-7179), ERK1/2 (sc-292838), p-Akt1/2/3 (sc-7985-R), Akt1/2/3 (sc-8312), VEGF (sc-152), and HIF-1α (sc-10790) were from Santa Cruz Biotechnology . Polyclonal antibody for p-JNK and JNK1/2 were from Cell Signaling Technology . PD98059, SP600125, LY294002 and apocynin were from Calbiochem . Monoclonal antibodies for β-actin, crystal violet, DCFH-DA and all other reagents of analytical grade as well as primers for RT-PCR were from Sigma Chemical Co , Boyden’s chambers were from Neuro Probe, Inc. . The monoclonal neutralizing antibody against Flk-1 was obtained from ImClone .n = 8 for any experimental group). Mice were fed as previously described [Methods: Animal experimentation. In this study we used three different dietary protocols for induction of progressive NAFLD that were carried out in 8 week old C57Bl/6 mice  (escribed  on the fn = 20) or from patients with simple steatosis (n = 10), referring to the Division of Gastroenterology and Hepatology of the University of Turin. All samples were collected at the time of first diagnosis; all subjects gave informed consent to the analysis, and the study protocol, which conformed to the ethical guidelines of the 1975 Declaration of Helsinki, was planned according to the guidelines of the local ethics committee.Human patients: The study on NASH patients was approved by the Ethics Committee of the Azienda Ospedaliera Universitaria Città della Salute . For this study we analyzed liver biopsies from NASH patients  the monoclonal antibody against OSM  or (ii) the monoclonal antibody against human CD68  or (iii) the secondary monoclonal antibody alone, as negative control. After blocking endogenous peroxidase activity with 3% hydrogen peroxide and performing microwave antigen retrieval in sodium citrate buffer pH6, primary antibodies were labeled by using EnVision, HRP-labeled System (DAKO) and visualized by 3′-diaminobenzidine substrate.Immunohistochemistry analysis: Liver sections from human patients with NASH or with simple steatosis were employed. Immunostaining procedure was as previously described . BrieflyLX2 cells culture: Human LX2 cells, a model of immortalized and activated, MF-like, human HSC, originally kindly provided by Prof. Scott L. Friedman , were cultured in Dulbecco’s modified Eagle’s medium , supplemented with 10% fetal calf serum and 1% antibiotics. In most experiments we also used human HSCs , were used between passages 4 and 7 when showing a phenotype of fully activated, MF-like HSCs (HSC/MFs), plated to obtain the desired sub-confluence level and then left for 24 h in serum-free Iscove’s medium to have cells at the lowest level of spontaneous proliferation . LX2 celCell migration and Chemotaxis: Non-oriented migration (chemokinesis) and chemotaxis of human LX2 (and HSC/MFs) were evaluated after exposure to PDGF-BB 10ng/mL, used as positive control, or to OSM 10 ng/mL, by performing the wound healing assay (20 h of incubation) or the modified Boyden’s chamber assay (6 h of incubation), as previously described ,13. For For the Boyden’s chamber assay, filter of 8 µm (Whatman Nuclepore™ track-etched polycarbonate membranes) were first coated with type I collagen (20 µg/mL) for 30 min at 37 °C.4 cells were placed in the upper compartment and were allowed to migrate through the pores of the filter into the lower compartment, in which chemotactic agents are present. At the end of incubation time (6 h), filters were removed from the Boyden’s chamber and stained with crystal violet. In some experiments, cells were pre-treated with specific inhibitors to check the relevance of specific proteins in process described.2 × 103 cells per well. The cells were incubated in serum-free medium (SFM) for 24 h and then exposed to OSM 10 ng/mL. At the desired time, the medium was removed, and the cells were washed twice with phosphate-buffered saline, fixed with 11% glutaraldehyde; after fixation, cells were washed and then stained with 0.1% (w/v) crystal violet solution for 10 min. After washing with water, the crystal violet was solubilized with 50 μL of 10% acetic acid solution, and absorbance was measured at 595–650 nm using a microplate reader . In some experiments, BrdU incorporation assay was performed. LX2 cells or HSC/MFs were seeded in culture plates , for 24 h up to 72 h. The cell proliferation rate was evaluated by means of a colorimetric assay kit supplied by Roche Diagnostic (11647229001) according to manufacturer’s instructions.Proliferation: Proliferation of human LX2 cells (and HSC/MFs) was evaluated by crystal violet proliferation assay by seeding cells in a 96-well plate at a density of 1.5 × 105 cells/well), were exposed to OSM 10 ng/mL for 15 min or pre-treated with apocynin for 1h and then exposed with OSM 10 ng/mL. ROS generation was detected as the conversion of 2′,7′-dichlorodihydrofluorescein diacetate  into the corresponding fluorescent derivative. Cells were observed and photographed under a Leica fluorescence microscope (DMI 4000 B model). (B) combination of DCFH-DA technique and flow cytometric analysis: cells were seeded in P35 dishes (5 × 105 cells/dish), cultured for 24 h and exposed to OSM alone or OSM plus 250 nM apocynin for 1 h before addition of 5 μM DCFH-DA (15 min of incubation in the dark). Cells were rapidly washed with PBS, collected by trypsinization, briefly centrifuged (1600 rpm for 5 min) and re-suspended in PBS for analysis. Detection of DCF green fluorescence (FL1) was performed on at least 5000 cells per sample with a FACScan equipped with a 488 nm argon laser using the CellQuest software . The peak of FL1 intensity of DCFH-DA-stained control cells grown without OSM was set to channel 101 and retained for all measurements [2O2 for 15 min, stained with DCFH-DA as above and assayed during the same analytical session.Detection of intracellular generation of ROS. (A) DCFH-DA technique: Detection of ROS generation in cultured cells was performed by using the semi-quantitative DCFH-DA technique as previously described . Culturew/v) non-fat dry milk and finally developed with the ECL reagents according to manufacturer’s instructions. Sample loading was evaluated by reblotting the same membrane with the un-phosphorylated form of protein or with β-actin antibody. In some experiments, protein levels in culture medium obtained from LX2 or HSC/MFs exposed to OSM for the desired time were evaluated by an immunoprecipitation procedure, as described previously [Western blot analysis: Total cell lysates, obtained as described ,11,24, weviously .® green method as described [PrimerSenseReversemurine OSMTTTCTCTGGGGATACCATCGGGAGACACGATGGGCTATGTmurine OSMβRGGAGACACGATGGGCTATGTCATCTGAGGTGATGGTGGTGmurine TBPCACATCACAGCTCCCCACCAAGCGGAGAAGATGCTGGAAAChuman OSMTACTGCTCACACAGAGGACGCCTATAGCCGCCATGCTCGChuman OSMRCGCGTCAGGTTTGCACTTA GTGTGTGGCACATTCCAAGhuman LIFRGGCTCATCACCACCTTCCAACCCCTTTCCCATCCCAACAAhuman HIF1αCCACCTATGACCTGCTTGGT TATCCAGGCTGTGTCGACTGhuman VEGFCCCACTGAGGAGTCCAACATTTTCTTGCGCTTTCGTTTTThuman CCL2 CCCCAGTCACCTGCTGTTAT AGATCTCCTTGGCCACAATGhuman IL6AGGAGACTTGCCTGGTGAAA CAGGGGTGGTTATTGCATCThuman TNF-αAACCTCCTCTCTGCCATCAA GGAAGACCCCTCCCAGATAGhuman GAPDHTGGTATCGTGGAGGACTCATGGAC ATGCCAGTGAGCTTCCCGTTCAGC Quantitative real-time PCR (Q-PCR): RNA extraction, complementary DNA synthesis, quantitative real-time PCR (Q-PCR) reactions were performed as previously described ,10. mRNAescribed . The ampTATA binding protein (TBP) and Gliceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as internal reference for murine and human sample respectively, and co-amplified with target samples using identical Q-PCR conditions. Samples were run in triplicate and mRNA expression was generated for each sample. Specificity of the amplified PCR products was determined by melting curve analysis and confirmed by agarose gel electrophoresis.p < 0.05 was considered significant).Statistical analysis: Data in bar graphs represent means ± SEM, and were obtained from average data of at least three independent experiments. Luminograms and morphological images are representative of at least three experiments with similar results. Statistical analysis for these experiments was performed by Student’s t-test or ANOVA for analysis of variance when appropriate  in order to evaluate selected pro-fibrogenic phenotypic responses of this cells. Human LX2 cells as well as HSC/MFs, that both positively responded to the potent mitogen and chemoattractant PDGF-BB (used here as positive control), did not proliferate when exposed to hrOSM according to data obtained in both the proliferation assay  and the The response to hrOSM was based, as expected, on the expression of both LIFβR e OSMβR receptor subunits in cultured cells; interestingly, treatment of LX2 and HSC/MFs cells with hrOSM also determined a significant increase of mRNA levels for both receptors that was detected 3 h after OSM administration . hrOSM, In both cell types OSM also induced an early  phosphorylation of ERK1/2 isoforms , JNK1/2 In order to elucidate which signaling pathway had a key role in modulating cell migration, both cell types were pre-treated with specific pharmacological inhibitors. Pre-treatment of cells with PD98059 (inhibitor of the ERK1/2 upstream kinase MEK-1) or SP600125 (inhibitor of the JNK1/2) or LY294002 (inhibitor of the Akt upstream kinase PI3K) significantly reduced either chemotaxis  or chemoSince literature data reported a role of OSM in triggering epithelial mesenchymal transition (EMT), we next performed experiments to evaluate a possible role of OSM in inducing EMT markers. However, in both cell types OSM was not able to affect typical EMT markers, like fibronectin and E-cadherin .In previous studies ,13,26 weApocynin also significantly inhibited OSM-induced chemotaxis in LX2 A,B and iIn order to further elucidate the role of ROS in mediating the chemotactic response, we performed more experiments by pre-treating LX2 with apocynin. Apocynin significantly inhibited early phosphorylation of ERK1/2 isoforms, JNK1/2 isoforms and c-Akt A,C,E, coSince in a previous study  we showeVEGF-A is a classic target gene of HIF1 (and then of HIF1α) and we previously demonstrated that this growth factor was able to stimulate migration of HSC/MFs . AccordiIn order to further investigate the relevance of OSM-dependent induction of VEGF-A expression, conditioned media obtained by cells exposed to hrOSM were collected after different time points (starting from 16 h to 48 h) and then used in the wound healing assay. OSM conditioned medium induced a significant stimulation of non-oriented migration at 48 h in both LX2 and HSC/MFs . Non-oriFinally, further preliminary experiments were performed in order to verify whether hrOSM, which is known to be mainly released by macrophages and lymphocytes, may also modulate the pro-inflammatory phenotypic response of LX2. The treatment of LX2 cells with hrOSM resulted in an increased transcription of CCL2, IL6, and TNFα A–C.Moreover, the pre-treatment of cells with apocynin or with AG490 reduced CCL2 transcription . The treMigration of hepatic MFs is a typical and distinctive pro-fibrogenic feature that allows these cells to align with inflammatory cells along fibrotic septa during liver fibrosis progression. This event occurs during CLDs in response to different stimuli and mediators, including polypeptide mediators, ROS as well as hypoxia ,7,11. InConcerning the putative pro-fibrogenic role of OSM, already proposed by two earlier studies ,22 and bA second role for OSM-related increase in intracellular ROS levels is related to the modulation of JAK/STAT signaling pathway. This event has been already described by Madamanchi et al. in vascular smooth muscle cells in which JAK2 was rapidly activated after treatment with hydrogen peroxide and both STAT1 and STAT3 were phosphorylated on tyrosine residues to then translocate to the nucleus in a JAK2-dependent manner . MoreoveResults obtained in the wound healing assay also provided evidence that OSM-dependent non oriented migration required late HIF1-dependent expression and release of VEGF-A. These results are homologous to those reported for exposure to hypoxic conditions and are potentially in agreement with literature data showing a role for OSM in modulating hypoxia-regulated processes , then suggesting a possible cross-talk between OSM and hypoxia signaling pathways . AccordiFinally, although in our experimental conditions OSM was found ineffective on the synthesis of collagen I and of TIMP1 (data not shown), differently from what reported by others , we addiIn conclusion, data reported in the present study provide novel evidence adding further knowledge on the proposed pro-fibrogenic role of OSM in the progression of CLD and suggest for the first time a possible involvement of this cytokine in the progression of NAFLD.