A Rapid and High‐Throughput Assay for the Estimation of Conversions of Ene‐Reductase‐Catalysed Reactions

A fast and sensitive colorimetric assay (FRED, fast and reliable ene‐reductases detection) that allows the estimation of levels of conversion of ene‐reductase (ER)‐catalysed reactions has been developed. The activated olefin is reduced by ER at the expense of NAD(P)H cofactor, whose regeneration is carried out in situ by the glucose/glucose dehydrogenase system. Subsequently, the consumption of the co‐substrate glucose is determined colorimetrically by a multienzymatic system. The FRED assay offers a wide range of possible applications, from enzyme fingerprinting and kinetic analysis, to primary screening of enzyme libraries and optimisation of ERs' performances under different reaction conditions.     

Catalytic activity and long-term stability of palladium oxide catalysts for natural gas combustion: Pd supported on LaMnO3-ZrO2

The catalytic activity and long-term stability of 2% Pd/LaMnO₃-ZrO₂ catalysts for natural gas combustion were deeply investigated. The catalyst, prepared via solution combustion synthesis, was completely characterized (XRD, BET, FESEM/EDS, TPC/TPD/TPR and FT-IR analysis) in the fresh status, and in the aged one, after prolonged treatment under hydro-thermal ageing and S-compounds poisoning (up to 3 weeks of hydro-thermal treatment at 800 °C under a flow of domestic boiler exhaust gases typical composition of 9% CO₂, 18% H₂O, 2% O₂ in N₂, including 200 ppmv of SO₂). An increased catalytic activity towards NG combustion with ageing was detected: the T₅₀, in fact, got lowered from 570 (fresh sample) to 465 °C (after 3 weeks ageing). Highly dispersed Pd centers were predominant on fresh catalyst. Upon ageing, oxygen covered Pd metal particles formed, at the expense of dispersed cationic and zerovalent Pd atoms. The increase in the catalytic activity was associated to the phase modification occurring in the bulk support, where Mn oxides, active towards CH₄ combustion, segregated. Moreover, bands due to sulfate species were detected in aged samples: IR analysis showed that Pd atoms did not interact significantly with these species. The bands of sulfate species decreased in intensity after 3 weeks ageing, likely mostly due to sintering of the catalyst, with the corresponding decrease in the surface area.     

Ewing’s Sarcoma: An Analysis of miRNA Expression Profiles and Target Genes in Paraffin-Embedded Primary Tumor Tissue

The molecular mechanism responsible for Ewing’s Sarcoma (ES) remains largely unknown. MicroRNAs (miRNAs), a class of small non-coding RNAs able to regulate gene expression, are deregulated in tumors and may serve as a tool for diagnosis and prediction. However, the status of miRNAs in ES has not yet been thoroughly investigated. This study compared global miRNAs expression in paraffin-embedded tumor tissue samples from 20 ES patients, affected by primary untreated tumors, with miRNAs expressed in normal human mesenchymal stromal cells (MSCs) by microarray analysis. A miRTarBase database was used to identify the predicted target genes for differentially expressed miRNAs. The miRNAs microarray analysis revealed distinct patterns of miRNAs expression between ES samples and normal MSCs. 58 of the 954 analyzed miRNAs were significantly differentially expressed in ES samples compared to MSCs. Moreover, the qRT-PCR analysis carried out on three selected miRNAs showed that miR-181b, miR-1915 and miR-1275 were significantly aberrantly regulated, confirming the microarray results. Bio-database analysis identified BCL-2 as a bona fide target gene of the miR-21, miR-181a, miR-181b, miR-29a, miR-29b, miR-497, miR-195, miR-let-7a, miR-34a and miR-1915. Using paraffin-embedded tissues from ES patients, this study has identified several potential target miRNAs and one gene that might be considered a novel critical biomarker for ES pathogenesis.     

Sugar‐Based Arylsulfonamide Carboxylates as Selective and Water‐Soluble Matrix Metalloproteinase‐12 Inhibitors

Matrix metalloproteinase‐12 (MMP‐12) can be considered an attractive target to study selective inhibitors useful in the development of new therapies for lung and cardiovascular diseases. In this study, a new series of arylsulfonamide carboxylates, with increased hydrophilicity resulting from conjugation with a β‐N‐acetyl‐d ‐glucosamine moiety, were designed and synthesized as MMP‐12 selective inhibitors. Their inhibitory activity was evaluated on human MMPs by using the fluorimetric assay, and a crystallographic analysis was performed to characterize their binding mode. Among these glycoconjugates, a nanomolar MMP‐12 inhibitor with improved water solubility, compound 3 [(R)‐2‐(N‐(2‐(3‐(2‐acetamido‐2‐deoxy‐β‐d ‐glucopyranosyl)thioureido)ethyl)biphenyl‐4‐ylsulfonamido)‐3‐methylbutanoic acid], was identified.     

Synthesis and Biological Evaluation of Pyrrolo[2,1‐f][1,2,4]triazine C‐Nucleosides with a Ribose, 2′‐Deoxyribose,and 2′,3′‐Dideoxyribose Sugar Moiety

The synthesis of hitherto unknown pyrrolo[2,1‐f][1,2,4]triazine C‐nucleosides is described. Structural variations (chlorine, bromine, iodine, and cyano groups) were introduced at position 7 of 4‐aza‐7,9‐dideazaadenine. In addition, pyrrolo[2,1‐f][1,2,4]triazine C‐nucleosides bearing a 2′‐deoxy‐, 2′,3′‐dideoxy‐, and 2′,3′‐dehydrodideoxyribose moiety were also prepared. Among these analogues, the pyrrolo[2,1‐f][1,2,4]triazine C‐ribonucleosides with either a hydrogen atom or cyano group at position 7 of the nucleobase displayed potent cytotoxic activity in a panel of various cancer cell lines.     

Potential of high pressure homogenization in the control and enhancement of proteolytic and fermentative activities of some Lactobacillus species

Different species of Lactobacillus involved in dairy product fermentation and ripening were considered in order to study the effect of high pressure homogenization (HPH) on: (i) fermentation kinetics of HPH treated cells inoculated in milk; (ii) metabolic profiles; (iii) release of intracellular proteolytic enzymes; and (iv) enhance of the activity of extracellular or cellular wall located proteolytic enzymes. The HPH treatments applied were 50, 100, 150 MPa, 2 cycles at 50 and at 100 MPa. The viability loss did not exceed 1.3 log cfu/ml after the higher treatments applied. The electrophoretic profiles of α- or β-casein incubated with the different cell free filtrates shown that HPH positively affected the proteolytic activity of some strains. Moreover, HPH affected the acidification rates of the milk inoculated with the processed cells and the primary metabolism of some strains. Regarding volatile compounds, ethanol, acetoin and 2-methyl butyric acid were subjected to the major changes when the inoculum had been processed.     

Avoiding cross-bifix-free binary words

In this paper we study the construction and the enumeration of binary words in $\{0,1\}^*$ having more 1’s than 0’s and avoiding a set of cross-bifix-free patterns. We give a particular succession rule, called jumping and marked succession rule, which describes the growth of such words according to their number of ones. Moreover, the problem of associating a word to a path in the generating tree obtained by the succession rule is solved by introducing an algorithm which constructs all binary words having more 1’s than 0’s and then kills those containing the forbidden patterns. Finally, we give the generating function of such words according to the number of ones.     

The Hedgehog-GLI Pathway Regulates MEK5-ERK5 Expression and Activation in Melanoma Cells

Malignant melanoma is the deadliest skin cancer, with a poor prognosis in advanced stages. We recently showed that the extracellular signal-regulated kinase 5 (ERK5), encoded by the MAPK7 gene, plays a pivotal role in melanoma by regulating cell functions necessary for tumour development, such as proliferation. Hedgehog-GLI signalling is constitutively active in melanoma and is required for proliferation. However, no data are available in literature about a possible interplay between Hedgehog-GLI and ERK5 pathways. Here, we show that hyperactivation of the Hedgehog-GLI pathway by genetic inhibition of the negative regulator Patched 1 increases the amount of ERK5 mRNA and protein. Chromatin immunoprecipitation showed that GLI1, the major downstream effector of Hedgehog-GLI signalling, binds to a functional non-canonical GLI consensus sequence at the MAPK7 promoter. Furthermore, we found that ERK5 is required for Hedgehog-GLI-dependent melanoma cell proliferation, and that the combination of GLI and ERK5 inhibitors is more effective than single treatments in reducing cell viability and colony formation ability in melanoma cells. Together, these findings led to the identification of a novel Hedgehog-GLI-ERK5 axis that regulates melanoma cell growth, and shed light on new functions of ERK5, paving the way for new therapeutic options in melanoma and other neoplasms with active Hedgehog-GLI and ERK5 pathways.     

Regenerative Strategies for Retinal Neurons: Novel Insights in Non-Mammalian Model Organisms

A detailed knowledge of the status of the retina in neurodegenerative conditions is a crucial point for the development of therapeutics in retinal pathologies and to translate eye research to CNS disease. In this context, manipulating signaling pathways that lead to neuronal regeneration offers an excellent opportunity to substitute damaged cells and, thus, restore the tissue functionality. Alternative systems and methods are increasingly being considered to replace/reduce in vivo approaches in the study of retina pathophysiology. Herein, we present recent data obtained from the zebrafish (Danio rerio) and the fruit fly Drosophila melanogaster that bring promising advantages into studying and modeling, at a preclinical level, neurodegeneration and regenerative approaches in retinal diseases. Indeed, the regenerative ability of vertebrate model zebrafish is particularly appealing. In addition, the fruit fly is ideal for regenerative studies due to its high degree of conservation with vertebrates and the broad spectrum of genetic variants achievable. Furthermore, a large part of the drosophila brain is dedicated to sight, thus offering the possibility of studying common mechanisms of the visual system and the brain at once. The knowledge acquired from these alternative models may help to investigate specific well-conserved factors of interest in human neuroregeneration after injuries or during pathologies.