Autoradiography of 3H-pirenzepine and 3H-AFDX-384 in Mouse Brain Regions: Possible Insights into M1, M2, and M4 Muscarinic Receptors Distribution.

Autoradiography helps to determine the distribution and density of muscarinic receptor (MR) binding sites in the brain. However, it relies on the selectivity of radioligands toward their target. 3H-Pirenzepine is commonly believed to label predominantly M1MR, 3H-AFDX-384 is considered as M2MR selective ligand. Here we performed series of autoradiographies with 3H-AFDX-384 (2 nM), and 3H-pirenzepine (5 nM) in WT, M1KO, M2KO, and M4KO mice to address the ligand selectivity. Labeling with 3H-pirenzepine using M1KO, M2KO, and M4KO brain sections showed the high selectivity toward M1MR. Selectivity of 3H-AFDX-384 toward M2MR varies among brain regions and depends on individual MR subtype proportion. All binding sites in the medulla oblongata and pons, correspond to M2MR. In caudate putamen, nucleus accumbens and olfactory tubercle, 77.7, 74.2, and 74.6% of 3H-AFDX-384 binding sites, respectively, are represented by M4MR and M2MR constitute only a minor portion. In cortex and hippocampus, 3H-AFDX-384 labels almost similar amounts of M2MR and M4MR alongside significant amounts of non-M2/non-M4MR. In cortex, the proportion of 3H-AFDX-384 binding sites attributable to M2MR can be increased by blocking M4MR with MT3 toxin without affecting non-M4MR. PD102807, which is considered as a highly selective M4MR antagonist failed to improve the discrimination of M2MR. Autoradiography with 3H-QNB showed genotype specific loss of binding sites. IN CONCLUSION: while 3H-pirenzepine showed the high selectivity toward M1MR, 3H-AFDX-384 binding sites represent different populations of MR subtypes in a brain-region-specific manner. This finding has to be taken into account when interpreting the binding data.

Comparison of analytical tools appropriate for identification of proteinaceous additives in historical mortars.

Natural organic additives such as eggs, lard, resins, and oils have been added to mortars since ancient times, because the ancient builders knew of their positive effect on the mortar quality. The tradition of adding organic materials to mortars was commonly handed down only verbally for thousands years. However, this practice disappeared in the nineteenth century, when the usage of modern materials started. Today, one of the most recent topics in the industry of building materials is the reusing of natural organic materials and searching for the forgotten ancient recipes. The research of the old technological approaches involves currently the most advanced analytical techniques and methods. This paper is focussed on testing the possibility of identification of proteinaceous additives in historical mortars and model mortar samples containing blood, bone glue, curd, eggs and gelatine, by Fourier transform infrared (FTIR) and Raman spectroscopy, gas chromatography - mass spectrometry (GC-MS), matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS), liquid chromatography-electrospray ionisation-quadrupole-time of flight mass spectrometry (LC-ESI-Q-TOF MS) and enzyme-linked immunosorbent assay (ELISA). All these methods were applied to the mortar sample taken from the interior of the medieval (sixteenth century) castle in Namest nad Oslavou in the Czech Republic and their comparison contributed to the rough estimation of the protein additive content in the mortar. The obtained results demonstrate that only LC-ESI-Q-TOF MS, MALDI-TOF MS and ELISA have the sufficiently low detection limits that enable the reliable identification of collagens in historical mortars. Graphical abstract Proteomics analyses of historical mortars.

A Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Method for the Identification of Anthraquinones: the Case of Historical Lakes.

This study deals with the identification of anthraquinoid molecular markers in standard dyes, reference lakes, and paint model systems using a micro-invasive and nondestructive technique such as matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry (MALDI-ToF-MS). Red anthraquinoid lakes, such as madder lake, carmine lake, and Indian lac, have been the most widely used for painting purposes since ancient times. From an analytical point of view, identifying lakes in paint samples is challenging and developing methods that maximize the information achievable minimizing the amount of sample needed is of paramount importance. The employed method was tested on less than 0.5 mg of reference samples and required a minimal sample preparation, entailing a hydrofluoric acid extraction. The method is fast and versatile because of the possibility to re-analyze the same sample (once it has been spotted on the steel plate), testing both positive and negative modes in a few minutes. The MALDI mass spectra collected in the two analysis modes were studied and compared with LDI and simulated mass spectra in order to highlight the peculiar behavior of the anthraquinones in the MALDI process. Both ionization modes were assessed for each species. The effect of the different paint binders on dye identification was also evaluated through the analyses of paint model systems. In the end, the method was successful in detecting madder lake in archeological samples from Greek wall paintings and on an Italian funerary clay vessel, demonstrating its capabilities to identify dyes in small amount of highly degraded samples. Graphical Abstract ᅟ.