Native extracellular matrix probes to target patient- and tissue-specific cell-microenvironment interactions by force spectroscopy

Atomic Force Microscopy (AFM) is successfully used for the quantitative investigation of the cellular mechanosensing of the microenvironment. To this purpose, several force spectroscopy approaches aim at measuring the adhesive forces between two living cells and also between a cell and an appropriate reproduction of the extracellular matrix (ECM), typically exploiting tips suitably functionalised with single components (e.g. collagen, fibronectin) of the ECM. However, these probes only poorly reproduce the complexity of the native cellular microenvironment and consequently of the biological interactions. We developed a novel approach to produce AFM probes that faithfully retain the structural and biochemical complexity of the ECM; this was achieved by attaching to an AFM cantilever a micrometric slice of native decellularised ECM, which was cut by laser microdissection. We demonstrate that these probes preserve the morphological, mechanical, and chemical heterogeneity of the ECM. Native ECM probes can be used in force spectroscopy experiments aimed at targeting cell-microenvironment interactions. Here, we demonstrate the feasibility of dissecting mechanotransductive cell-ECM interactions in the 10 pN range. As proof-of-principle, we tested a rat bladder ECM probe against the AY-27 rat bladder cancer cell line. On the one hand, we obtained reproducible results using different probes derived from the same ECM regions; on the other hand, we detected differences in the adhesion patterns of distinct bladder ECM regions (submucosa, detrusor, and adventitia), in line with the disparities in composition and biophysical properties of these ECM regions. Our results demonstrate that native ECM probes, produced from patient-specific regions of organs and tissues, can be used to investigate cell-microenvironment interactions and early mechanotransductive processes by force spectroscopy. This opens new possibilities in the field of personalised medicine

Ruthenacycles and Iridacycles as Catalysts for Asymmetric Transfer Hydrogenation and Racemisation

Ruthenacycles, which are easily prepared in a single step by reaction between enantiopure aromatic amines and [Ru(arene)Cl2]2 in the presence of NaOH and KPF6, are very good asymmetric transfer hydrogenation catalysts. A range of aromatic ketones were reduced using isopropanol in good yields with ee’s up to 98%. Iridacycles, which are prepared in similar fashion from [IrCp*Cl2]2 are excellent catalysts for the racemisation of secondary alcohols and chlorohydrins at room temperature. This allowed the development of a new dynamic kinetic resolution of chlorohydrins to the enantiopure epoxides in up to 90% yield and 98% enantiomeric excess (ee) using a mutant of the enzyme Haloalcohol dehalogenase C and an iridacycle as racemisation catalyst.

Mechanistic and Kinetic Investigation on the Formation of Palladacyclopentadiene Complexes. A Novel Interpretation Involving a Bimolecular Self Reaction of a Monoalkyne Intermediate

The stoichiometric reaction between the complex [Pd(eta(2)-dmfu)(BiPy)] (dmfu = dimethylfumarate; BiPy = 2,2'-bipyridine) and the deactivated alkynes dmbd (dimethyl-2-butynedioate) and pna (methyl (4-nitrophenyl)propynoate), providing the respective palladacyclopentadienes, was investigated. The mechanism leading to the palladacyclopentadiene derivative involves a bimolecular self-rearrangement of the monoalkyne intermediate [Pd(eta(2)-alk)(BiPy)] (alk = dmbd, pna), followed by the customary attack of the free alkyne on the intermediate [Pd(eta(2)-alk)(BiPy)] itself and on the elusive and highly reactive "naked palladium" [Pd(BiPy)(0)] formed. The alkyne pna proved to be less effective in the displacement of dmfu than dmbd. The reaction under stoichiometric equimolar conditions of the latter with [Pd(eta(2)-dmfu)(BiPy)] allows the direct determination of the bimolecular self-reaction rate constant k(c) and consequently the assessment of all the rate constants involved in the overall mechanistic network

Immediate early transcription activation by salicylic acid via the cauliflower mosaic virus as-1 element.

Transgenic tobacco plants carrying a number of regulatory sequences derived from the cauliflower mosaic virus 35S promoter were tested for their response to treatment with salicylic acid (SA), an endogenous signal involved in plant defense responses. beta-Glucuronidase (GUS) gene fusions with the full-length (-343 to +8) 35S promoter or the -90 truncation were found to be induced by SA. Time course experiments revealed that, in the continuous presence of SA, the -90 promoter construct (-90 35S-GUS) displayed rapid and transient induction kinetics, with maximum RNA levels at 1 to 4 hr, which declined to low levels by 24 hr. Induction was still apparent in the presence of the protein synthesis inhibitor cycloheximide (CHX). Moreover, mRNA levels continued to accumulate over 24 hr rather than to decline. By contrast, mRNA from the endogenous pathogenesis-related protein-1a (PR-1a) gene began to accumulate at later times during SA treatment and steadily increased through 24 hr; transcription of this gene was almost completely blocked by the presence of CHX. Further dissection of the region from -90 and -46 of the 35S promoter revealed that the SA-responsive element corresponds to the previously characterized activation sequence-1 (as-1). These results represent a definitive analysis of immediate early responses to SA, relative to the late induction of PR genes, and potentially elucidate the early events of SA signal transduction during the plant defense response

Identification of NPR1-dependent and independent genes early induced by salicylic acid treatment in Arabidopsis

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Subtle balance of steric and electronic effects for the synthesis of atactic polyketones catalyzed by Pd complexes with meta-substituted aryl-BIAN ligands

Both symmetric and nonsymmetric bis(aryl)acenaphthenequinonediimine ligands, featured by substituents in meta-positions of the aryl rings, have been applied for the first time as ancillary ligands for the palladium-catalyzed CO/vinyl arene copolymerization. The nature and the number of substituents affect both the productivity and the molecular weight of the synthesized copolymers. Palladium complexes containing the nonsymmetric ligands are the most efficient catalysts reported so far for the synthesis of atactic polyketones

Subtle Balance of Steric and Electronic Effects for the Synthesis of Atactic Polyketones Catalyzed by Pd-Complexes with meta-Substituted Aryl-BIAN Ligands

Both symmetric and nonsymmetric bis(aryl)acenaphthenequinonediimine ligands, featured by substiuents in meta-positions of the aryl rings, have been applied for the first time as ancillary ligands for the palladium-catalyzed CO/vinyl arene copolymerization. The nature and the number of substituents affect both the productivity and the molecular weight of the synthesized copolymers. Palladium complexes containing the nonsymmetric ligands are the most efficient catalysts reported so far for the synthesis of atactic polyketones