Stereoselective Synthesis of α,α’-Dihydroxy-β,β’-diaryl-β-amino Acids by Mannich-like Condensation of Hydroarylamides

Dual \u3b1,\u3b1\u2019-Dihydroxy-\u3b2-amino acids are very interesting tools for several industrial applications. Nevertheless, few derivatives are reported in the literature concerning the substitution pattern as well as their enantioselective syntheses are lacking. Here, we report on the preparation of enantiopure \u3b1,\u3b1\u2019-dihydroxy-\u3b2,\u3b2\u2019-diaryl-\u3b2-amino acid (dual) derivatives by an efficient Mannich-like condensation of hydroarylamides with 5,6-diethoxy-5,6-dimethyl-1,4-dioxan-2-one (triethylsilyl)ketene acetal. The synthetic protocol has been optimized affording the dual compounds in very good yields and with different aryl substitution patterns. Taking advantage of the \u201cdouble stereodifferentiation\u201d concept, a highly stereoselective reaction was performed: of the 16 possible isomers, only two diastereoisomers (d.r. up to 93:7) formed. Insights on the high stereocontrol of this condensation were given

Retinoblastoma Is Characterized by a Cold, CD8+ Cell Poor, PD-L1- Microenvironment, Which Turns Into Hot, CD8+ Cell Rich, PD-L1+ After Chemotherapy

PURPOSE. To investigate the impact of chemotherapy (CHT) on human retinoblastoma (RB) tumor microenvironment (TME).CASES AND METHODS. Ninety-four RBs were studied, including 44 primary RBs treated by upfront surgery (Group 1) and 50 primary RBs enucleated after CHT (CHT), either intraarterial (IAC; Group 2, 33 cases) or systemic (S-CHT; Group 3, 17 cases). Conventional and multiplexed immunohistochemistry were performed to make quantitative comparisons among the three groups, for the following parameters: tumor-infiltrating inflammatory cells (TI-ICs); programmed cell death protein 1 (PD-1) positive TI-ICs; Ki67 proliferation index; gliosis; PD-1 ligand (PD-L1) protein expression; vessel number. We also correlated these TME factors with the presence of histological high-risk factors (HHRF+) and RB anaplasia grade (AG).RESULTS. After CHT, a decrease in both RB burden and Ki67 positivity was observed. In parallel, most subsets of TI-ICs, PD-1+ TI-ICs, gliosis, and PD-L1 protein expression significantly increased (P < 0.001, P = 0.02, P < 0.001, respectively). Vessel number did not significantly vary. Age, HHRFs+ and AG were significantly different between primary and chemoreduced RBs (P < 0.001, P = 0.006, P = 0.001, respectively) and were correlated with most TME factors.CONCLUSIONS. CHT modulates host antitumor immunity by reorienting the RB TME from anergic into an active, CD8+, PD-L1+ hot state. Furthermore, some clinicopathological characteristics of RB correlate with several factors of TME. Our study adds data in favor of the possibility of a new therapeutic scenario in human RB

Novel compounds targeting PFKFB3, the key glycolytic enzyme, as a way to inhibit angiogenesis

Angiogenesis is an important contributor to atherosclerotic plaque growth and instability. Clinical evidence has linked intraplaque angiogenesis with progressive and unstable vascular disease. Proliferating endothelial cells (ECs) can switch their metabolism to being highly glycolytic enabling their growth and division in the angiogenic process. Recent studies have demonstrated the therapeutic potential of 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), a commercially available 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) inhibitor, in angiogenesis models. For this reason, PFKFB3 inhibitors seem promising compounds to be used in promoting plaque stability. In the present study, we studied the in vitro effects of the PFKFB3 inhibitor 3PO, and of two self-synthesized inhibitors named phenoxindazole analogues (PA-1 and PA-2; based on Boyd et al., 2015) on glycolysis, cell proliferation, migration, matrix metalloproteinase (MMP) activity and gene expression in ECs. We observed that these compounds were able to significantly reduce glycolysis levels in the human endothelial cell line EA.hy926. In addition, all three compounds markedly reduced endothelial cell migration, proliferation and wound closing capacity which are essential for neovessel formation. Moreover, we demonstrated by gelation gel zymography that these inhibitors reduce the activity of proMMP-9 and MMP-2 up to 40-50% and 20-30% compared to control, respectively. Furthermore, real-time PCR results indicate that the PA compounds downregulate PFKFB3 gene expression whilst 3PO does not. As for markers of migration and angiogenesis, such as ICAM and VEGFR2, these were markedly reduced. Finally, gelatinase gene expression was downregulated by up to 80%. These findings show that PFKFB3 inhibition with PA compounds markedly reduce endothelial cell migration, proliferation and gelatinolytic activity concomitant with a significant decrease in gelatinase gene expression, EC migration and angiogenesis markers. Thus, these compounds have the potential to be tested in an animal model of angiogenesis. This project has been funded by the European Union\u2019s Horizon 2020 Marie Sk\u142odowska-Curie grant (#67552)

Rational design of a user-friendly aptamer/peptide-based device for the detection of staphylococcus aureus

The urgent need to develop a detection system for Staphylococcus aureus, one of the most common causes of infection, is prompting research towards novel approaches and devices, with a particular focus on point-of-care analysis. Biosensors are promising systems to achieve this aim. We coupled the selectivity and affinity of aptamers, short nucleic acids sequences able to recognize specific epitopes on bacterial surface, immobilized at high density on a nanostructured zirconium dioxide surface, with the rational design of specifically interacting fluorescent peptides to assemble an easy-to-use detection device. We show that the displacement of fluorescent peptides upon the competitive binding of S. aureus to immobilized aptamers can be detected and quantified through fluorescence loss. This approach could be also applied to the detection of other bacterial species once aptamers interacting with specific antigens will be identified, allowing the development of a platform for easy detection of a pathogen without requiring access to a healthcare environment

Angiogenesis inhibition with novel compounds targeting the key glycolytic enzyme PFKFB3

Aim Intraplaque angiogenesis is associated with progressive and unstable vascular disease. Angiogenesis signals induce endothelial cells (ECs) to switch their metabolism to being highly glycolytic enabling their growth and division. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) is a key glycolytic activator. PFKFB3 inhibitors have been shown to reduce EC sprouting and seem promising compounds to be used in promoting plaque stability. Methods We studied in the human EC line EA.hy926 the effects of 3PO, a commercially available inhibitor of PFKFB3, and of two self-synthesized phenoxindazole analogues (PA-1 and PA-2; based on Boyd et al., 2015) on glycolysis, proliferation, migration, capillary networking, matrix metalloproteinase (MMP) activity and gene expression. Results The three compounds significantly reduced glycolysis levels, while PA-1 and PA-2 affected also capillary-like structure networking. Accordingly, the compounds markedly inhibited EC migration, proliferation and wound closing capacity which are essential for neovessel formation. Moreover, these inhibitors reduced the activity of proMMP-9 and MMP-2 up to 40-50% and 20-30% compared to control, respectively, while gelatinase genes expression was downregulated by 80%. Finally, the PA compounds downregulated PFKFB3 expression whilst 3PO did not. Similarly, markers of migration and angiogenesis, such as CCL5, VCAM, VEGFA and VEGFR2, were also markedly reduced (up to 10-fold). Conclusions These findings show that PFKFB3 inhibition with PA compounds interferes with key pro-angiogenic functions, such as endothelial migration, proliferation and capillary-like structure and molecularly exerts a multitarget anti-angiogenetic activity. This project has been funded by the European Union\u2019s Horizon 2020 Marie Sklodowska Curie grant (#67552)

Inhibition of the Key Glycolytic Enzyme PFKFB3 with Novel Compounds Suppresses Vessel Sprouting

Aim Intraplaque angiogenesis is an important contributor to atherosclerotic plaque growth and instability. Angiogenic signals induce endothelial cells (ECs) to switch their metabolism to being highly glycolytic, enabling their growth and division. Glycolytic modulation by inhibition of the glycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) has been shown to reduce angiogenesis. The objective of this study was to identify novel anti-angiogenic compounds with a potential to efficiently modulate (inhibit) angiogenesis. Methods Using the human EC line EA.hy926, we studied the effects of PFKFB3 inhibition with 3PO, a weak competitive inhibitor of PFKFB3, and of two potent self-synthesized phenoxindazole analogues (PA-1 and PA-2) on glycolysis, proliferation, migration, matrix metalloproteinase (MMP) activity, and capillary tube formation. The latter experiment was also performed using primary human umbilical vein endothelial cells (HUVEC). Moreover, gene expression of important markers related to angiogenesis were measured at mRNA level by real-time PCR. Results PFKFB3 inhibition with all three tested compounds significantly reduced glycolytic activity. While PA-1 and PA-2 suppressed capillary tube formation in both cell groups, 3PO did not have any effect in EA.hy926 ECs and even an inducing effect in the HUVECs. Accordingly, PA-1 and PA-2 markedly inhibited EC migration, proliferation and wound closing capacity which are essential for neovessel formation. Moreover, these inhibitors downregulated gelatinase gene expression up to 6-fold, as well reduced the activity of proMMP-9 and MMP-2 up to 50% and 30% compared to control, respectively. Gene expression analysis revealed that the PA compounds downregulated PFKFB3 expression whilst 3PO did not. Similarly, markers of migration and angiogenesis, such as CCL5, VCAM-1, VEGFA and VEGFR2, were also markedly reduced (up to 10-fold) by the PA compounds. Conclusions These findings suggest that PFKFB3 inhibition with PA compounds may interfere with key pro-angiogenic functions, such as endothelial migration, proliferation and capillary-like structure formation and this exerts a multitarget anti-angiogenic activity. Hence, PFKFB3 inhibition with PA compounds is a promising therapeutic approach to promote plaque stability

Novel MMP-inhibiting peptides for stabilizing atherosclerotic plaques

Aim: Matrix-degrading metalloproteases (MMPs) play an essential role in the atherogenic process, from the initial lesion to plaque rupture. A growing body of evidence show that inhibition of MMP activity or rebalancing the MMP/TIMP (tissue inhibitor of MMP) equilibrium has a potential therapeutic strategy for atherosclerosis. We studied the in vitro effects of newly designed peptides on MMP activity in endothelial cells and macrophages. Methods: Eight peptides were designed in silico and synthesized using the solid phase synthesis and characterized by circular dichroism and Dynamic Light Scattering. The mouse macrophage cell line J774A.1 and the human endothelial cell line EA.hy926 were treated with the peptides (10 \ub5\uf06dM and 10 \ub5\uf06dM) for 24 hours. Then, conditioned medium was collected and MMP-2, and MMP-9 activity was determined by gelatin gel zymography. Results: Analysis of the zymograms showed that upon 10 \ub5M treatment in endothelial cells, three out of eight peptides reduced the activity of proMMP-9 and proMMP-2 by more than 60 and 40 percent, respectively. Whereas, upon treatment at 100 \ub5M the proMMP-9 levels were less or not reduced. These could be consequent to aggregation of the peptide at a concentration higher than 50 \ub5\uf06dM. In macrophages, two of these peptides reduced the activity of proMMP-2 by more than 40 percent, but had no effect on proMMP-9 activity. The peptides, did not show any effects on MMP mRNA levels. Conclusions: These data show that these peptides show a promising inhibitory effect on MMP-9 and MMP-2 activity in endothelial cells and macrophages

Improved calibration of a solid substrate fermentation model

Background: Calibration of dynamic models in biotechnology is challenging. Kinetic models are usually complex and differential equations are highly coupled involving a large number of parameters. In addition, available measurements are scarce and infrequent, and some key variables are often non-measurable. Therefore, effective optimization and statistical analysis methods are crucial to achieve meaningful results. In this research, we apply a metaheuristic scatter search algorithm to calibrate a solid substrate cultivation model. Results: Even though scatter search has shown to be effective for calibrating difficult nonlinear models, we show here that a posteriori analysis can significantly improve the accuracy and reliability of the estimation. Conclusions: Sensibility and correlation analysis helped us detect reliability problems and provided suggestions to improve the design of future experiments

Electrospun aniline-tetramer-co-polycaprolactone fibers for conductive, biodegradable scaffolds

Conjugated polymers have been proposed as promising materials for scaffolds in tissue engineering applications. However, the restricted processability and biodegradability of conjugated polymers limit their use for biomedical applications. Here we synthesized a block-co-polymer of aniline tetramer and PCL (AT-PCL), and processed it into fibrous non-woven scaffolds by electrospinning. We showed that fibronectin (Fn) adhesion was dependent on the AT-PCL oxidative state, with a reduced Fn unfolding length on doped membranes. Furthermore, we demonstrated the cytocompatibility and potential of these membranes to support the growth and osteogenic differentiation of MC3T3-E1 cells over 21 days