The Fibrin Matrix Regulates Angiogenic Responses within the Hemostatic Microenvironment through Biochemical Control

Conceptually, premature initiation of post-wound angiogenesis could interfere with hemostasis, as it relies on fibrinolysis. The mechanisms facilitating orchestration of these events remain poorly understood, however, likely due to limitations in discerning the individual contribution of cells and extracellular matrix. Here, we designed an in vitro Hemostatic-Components-Model (HCM) to investigate the role of the fibrin matrix as protein factor-carrier, independent of its cell-scaffold function. After characterizing the proteomic profile of HCM-harvested matrix releasates, we demonstrate that the key pro-/anti-angiogenic factors, VEGF and PF4, are differentially bound by the matrix. Changing matrix fibrin mass consequently alters the balance of releasate factor concentrations, with differential effects on basic endothelial cell (EC) behaviors. While increasing mass, and releasate VEGF levels, promoted EC chemotactic migration, it progressively inhibited tube formation, a response that was dependent on PF4. These results indicate that the clot’s matrix component initially serves as biochemical anti-angiogenic barrier, suggesting that post-hemostatic angiogenesis follows fibrinolysis-mediated angiogenic disinhibition. Beyond their significance towards understanding the spatiotemporal regulation of wound healing, our findings could inform the study of other pathophysiological processes in which coagulation and angiogenesis are prominent features, such as cardiovascular and malignant disease.     

Investigation of the efficacy of paclitaxel on some miRNAs profiles in breast cancer stem cells

Understanding of the functions of microRNAs in breast cancer and breast cancer stem cells have been a hope for the development of new molecular targeted therapies. Here, it is aimed to investigate the differences in the expression levels of let-7a, miR-10b, miR-21, miR-125b, miR-145, miR-155, miR-200c, miR-221, miR-222 and miR-335, which associated with gene and proteins in MCF-7 (parental) and MCF-7s (Mammosphere/stem cell-enriched population/CD44+/CD24-cells) cells treated with paclitaxel. MCF-7s were obtained from parental MCF-7 cells. Cytotoxic activity of paclitaxel was determined by ATP assay. Total RNA isolation and cDNA conversion were performed from the samples. Changes in expression levels of miRNAs were examined by RT-qPCR. Identified target genes and proteins of miRNAs were analyzed with RT-qPCR and western blot analysis, respectively. miR-125b was significantly expressed (2.0946-fold; p = 0.021) in MCF-7s cells compared to control after treatment with paclitaxel. Downregulation of SMO, STAT3, NANOG, OCT4, SOX2, ERBB2 and ERBB3 and upregulation of TP53 genes were significant after 48 h treatment in MCF-7s cells. Protein expressions of SOX2, OCT4, SMAD4, SOX2 and OCT4 also decreased. Paclitaxel induces miR-125b expression in MCF-7s cells. Upregulation of miR-125b may be used as a biomarker for the prediction of response to paclitaxel treatment in breast cancer.     

Interleukin-33 Is Biologically Active Independently of Caspase-1 Cleavage

The new interleukin (IL)-1 family cytokine IL-33 is synthesized as a 30-kDa precursor. Like pro-IL-1β, human pro-IL-33 was reported to be cleaved by caspase-1 to generate an 18-kDa fragment, which is sufficient to activate signaling by the IL-33 receptor T1/ST2. However, the proposed caspase-1 cleavage site is poorly conserved between species. In addition, it is not clear whether caspase-1 cleavage of pro-IL-33 occurs in vivo and whether, as for IL-1β, this cleavage is a prerequisite for IL-33 secretion and bioactivity. In this study, we further investigated caspase-1 cleavage of mouse and human pro-IL-33 and assessed the potential bioactivity of the IL-33 precursor. We observed the generation of a 20-kDa IL-33 fragment in cell lysates, which was enhanced by incubation with caspase-1. However, in vitro assays of mouse and human pro-IL-33 indicated that IL-33 is not a direct substrate for this enzyme. Consistently, caspase-1 activation in THP-1 cells induced cleavage of pro-IL-1β but not of pro-IL-33, and activated THP-1 cells released full-length pro-IL-33 into culture supernatants. Finally, addition of full-length pro-IL-33 induced T1/ST2-dependent IL-6 secretion in mast cells. However, we observed in situ processing of pro-IL-33 in mast cell cultures, and it remains to be determined whether full-length pro-IL-33 itself indeed represents the bioactive species. In conclusion, our data indicate that pro-IL-33 is not a direct substrate for caspase-1. In addition, our results clearly show that caspase-1 cleavage is not required for pro-IL-33 secretion and bioactivity, highlighting major differences between IL-1β and IL-33.Interleukin (IL)² -33, the most recently described cytokine of the IL-1 family, is synthesized as a 30-kDa precursor. Human pro-IL-33, like pro-IL-1β, was reported to be cleaved by caspase-1 in vitro to generate an 18-kDa fragment, termed mature IL-33, which is sufficient to activate signaling by the IL-33 receptor T1/ST2 (1).Caspase-1 is an endoproteinase that specifically cleaves Asp-Xaa bonds, where Xaa typically refers to a small, often hydrophobic residue (2–4). Caspase-1 activity absolutely requires the presence of an Asp residue at position −1 of the cleavage site. Consistently, replacement of Asp¹¹⁶ by other amino acids, such as Ala, was previously demonstrated to prevent caspase-1 cleavage of pro-IL-1β (2). Recombinant (r) mature IL-33 starts at Ser¹¹² for human (h) IL-33 and at Ser¹⁰⁹ for mouse (m) IL-33, neither of which corresponds exactly to the position of a potential caspase-1 cleavage site. Indeed, the N-terminal moiety of human pro-IL-33 sequence contains a single Asp at position 110, and the N-terminal portion of mouse pro-IL-33 contains an Asp at positions 88 and 106. In fact, the region located between amino acids 80 and 110 of pro-IL-33 is rather poorly conserved between species (5). In particular, no Asp residues can be consistently found at an identical position across species to hint at the presence of a conserved caspase-1 cleavage site. So far, caspase-1 cleavage of pro-IL-33 has not been investigated in any species other than human.Expression of endogenous IL-33 has been described most extensively in endothelial cells, where essentially nuclear, full-length 30-kDa pro-IL-33 is detected (5–7). To date, only two studies have examined potential effects of caspase-1 activation on the processing and secretion of pro-IL-33 in living cells. In one study, stimulation of murine glial cultures with caspase-1 activators induced secretion of bioactive IL-33 into culture supernatants, but the size of the secreted protein was not assessed (8). It is thus not clear whether caspase-1 cleavage of pro-IL-33 occurs in mouse cells. In a second study, Western blot analysis revealed the presence of a 32-kDa protein and minor 17 and 20 kDa bands reacting with anti-IL-33 antibodies in the supernatants of THP-1 cells upon caspase-1 activation, suggesting secretion of full-length pro-IL-33 and of two potential cleavage products (9). Although this last observation suggests that some pro-IL-33 may be secreted, it not known to what extent IL-33 secretion is dependent on caspase-1 cleavage. Finally, so far all studies reporting T1/ST2-mediated effects of IL-33 were performed using the recombinant mature form of IL-33, whereas potential biological activity of the full-length precursor form has not been tested. It thus remains to be shown whether, as for IL-1β, caspase-1 cleavage is indeed required for IL-33 bioactivity. In the present study, we thus further investigated caspase-1 cleavage of mouse and human pro-IL-33 in vitro and in cultured cells and assessed the potential bioactivity of the IL-33 precursor.     

Structure/Function Analysis of PARP-1 in Oxidative and Nitrosative Stress-Induced Monomeric ADPR Formation

Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2^nd messenger monomeric adenosine diphosphate-ribose (mADPR). Previous studies have delineated specific roles for several of PARP-1′s structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1′s BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2^nd messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.     

Nonsecreted insulin-like growth factor binding protein-3 (IGFBP-3) can induce apoptosis in human prostate cancer cells by IGF-independent mechanisms without being concentrated in the nucleus

Insulin-like growth factor binding protein-3 (IGFBP-3), a secreted protein, has the intrinsic ability to induce apoptosis directly without binding insulin-like growth factors. Previous studies suggested that IGFBP-3 must be secreted to exert its biological functions. IGFBP-3 contains a nuclear localization signal (NLS), and exogenous IGFBP-3 is translocated into the nucleus, suggesting that both secretion and nuclear localization may play important roles in IGFBP-3 action. To address these questions, we fused yellow fluorescent protein (YFP) to mature IGFBP-3 lacking its signal peptide so that it would remain intracellular and mutated the C-terminal NLS of IGFBP-3, (228)KGRKR(232), to MDGEA. Following transfection of PC-3 human prostate cancer cells with these constructs, Western blots indicated that YFP-IGFBP-3 lacking a signal peptide was cell-associated and not present in the extracellular media. Moreover, the fusion protein was not N-glycosylated, indicating that it had not entered the secretory pathway. Confocal imaging showed that intracellular YFP-MDGEA-IGFBP-3 was predominantly cytoplasmic. Transient transfection of nonsecreted YFP-wild-type IGFBP-3 decreased cell viability, as assessed by staining with annexin V followed by flow cytometry. Induction of cell death was caspase-dependent, indicative of apoptosis. Apoptosis also was induced by the nonsecreted NLS mutant (YFP-MDGEA-IGFBP-3) alone and when the IGF-binding site also had been mutated. These results indicate that IGFBP-3 can induce apoptosis in an IGF-independent manner without being secreted or concentrated in the nucleus.     

Prediction of optimal folding routes of proteins that satisfy the principle of lowest entropy loss: dynamic contact maps and optimal control

An optimization model is introduced in which proteins try to evade high energy regions of the folding landscape, and prefer low entropy loss routes during folding. We make use of the framework of optimal control whose convenient solution provides practical and useful insight into the sequence of events during folding. We assume that the native state is available. As the protein folds, it makes different set of contacts at different folding steps. The dynamic contact map is constructed from these contacts. The topology of the dynamic contact map changes during the course of folding and this information is utilized in the dynamic optimization model. The solution is obtained using the optimal control theory. We show that the optimal solution can be cast into the form of a Gaussian Network that governs the optimal folding dynamics. Simulation results on three examples (CI2, Sso7d and Villin) show that folding starts by the formation of local clusters. Non-local clusters generally require the formation of several local clusters. Non-local clusters form cooperatively and not sequentially. We also observe that the optimal controller prefers "zipping" or small loop closure steps during folding. The folding routes predicted by the proposed method bear strong resemblance to the results in the literature.     

Root hairs are the most important root trait for rhizosheath formation of barley (Hordeum vulgare), maize (Zea mays) and Lotus japonicus (Gifu)

Background and AimsRhizosheaths are defined as the soil adhering to the root system after it is extracted from the ground. Root hairs and mucilage (root exudates) are key root traits involved in rhizosheath formation, but to better understand the mechanisms involved their relative contributions should be distinguished.MethodsThe ability of three species [barley (Hordeum vulgare), maize (Zea mays) and Lotus japonicus (Gifu)] to form a rhizosheath in a sandy loam soil was compared with that of their root-hairless mutants [bald root barley (brb), maize root hairless 3 (rth3) and root hairless 1 (Ljrhl1)]. Root hair traits (length and density) of wild-type (WT) barley and maize were compared along with exudate adhesiveness of both barley and maize genotypes. Furthermore, root hair traits and exudate adhesiveness from different root types (axile versus lateral) were compared within the cereal species.Key ResultsPer unit root length, rhizosheath size diminished in the order of barley > L. japonicus > maize in WT plants. Root hairs significantly increased rhizosheath formation of all species (3.9-, 3.2- and 1.8-fold for barley, L. japonicus and maize, respectively) but there was no consistent genotypic effect on exudate adhesiveness in the cereals. While brb exudates were more and rth3 exudates were less adhesive than their respective WTs, maize rth3 bound more soil than barley brb. Although both maize genotypes produced significantly more adhesive exudate than the barley genotypes, root hair development of WT barley was more extensive than that of WT maize. Thus, the greater density of longer root hairs in WT barley bound more soil than WT maize. Root type did not seem to affect rhizosheath formation, unless these types differed in root length.ConclusionsWhen root hairs were present, greater root hair development better facilitated rhizosheath formation than root exudate adhesiveness. However, when root hairs were absent root exudate adhesiveness was a more dominant trait.     

The role of the ADRA2A C1291G genetic polymorphism in response to dexmedetomidine on patients undergoing coronary artery surgery

The existence of interindividual drug response variability has been known for a long time. Individual susceptibility which might cause toxicity or inadequate treatment is important in drug therapy. Genetic polymorphisms in genes responsible for drug response are expected to be useful in keeping track of differences among individuals. Dexmedetomidine is a sedative drug, whose use in intensive care unit patients was confirmed by USA-Food Drug Administration (FDA) by the end of 1999. It was proven that dexmedetomidine shows its clinic effect via the α₂-AR. However, to the best of our knowledge, to date, there is no investigation in clinic indicating the relation between dexmedetomidine and α_2A-AR gene polymorphism. The aim of our study was to investigate the association between the effect of α_2A-Adrenergic Receptor (ADRA2A) C-1291G gene polymorphism in the promoter region of the candidate gene and clinical effects (sedative and haemodynamics effects) of dexmedetomidine. One hundred and ten patients undergoing coronary artery surgery were prospectively studied. Anesthetic technique was standardized with fentanyl, midazolam and rocuronium bromide. Patients were monitorized with Bispectral Index (BIS) monitor in addition to the routine invasive haemodynamic monitorization in the operation room. The Ramsay Sedation Scale was also used in order to determine the sedation level just arriving to Intensive Care Unit (ICU). The genotyping of ADRA2A C1291G was done by Restriction Fragment Length Polymorphism-Polymerase Chain Reaction (RFLP-PCR). We found the frequencies of C1291C, C1291G and G1291G genotypes, as 43.6, 45.5 and 10.9%, respectively. Patients who carry variant genotype had higher BIS and Ramsay Sedation Scores, indicating a longer period for falling asleep. The results of our study are promising, considering the association between ADRA2A polymorphism and response to dexmedetomidine. However, further investigations on other ADRA2A locus or haplotypes might be useful to clarify the relation between this gene and dexmedetomidine activity.