What Sherlock sorely missed: the EVA technology for cultural heritage exploration.

Introduction: Capture of proteins and metabolites from Cultural Heritage (paintings, manuscripts, parchment etc.) has been done in the past via surface scraping and erasing, a method discouraged. The EVA (ethylene vinyl acetate) method consists of a plastic polymer in which strong cation and anion resins, admixed with C8 and/or C18, are embedded. Areas covered: We review here the findings on different items stored in public libraries and archives: (a) the original manuscript of the novel Master y Margarita by Bulgakov; (b) the death registries of the lazaretto in the 1630 Milano plague; (c) the shirt worn by A. Chekhov in his death bed; (d) Kepler's script on Hipparchus (in St. Petersburg National Archives); (e) the Memoirs of G. Casanova. Expert opinion: The technique here surveyed appears to be a unique tool enabling exploration of any document stored in public archives, museum and private collections without damaging or contaminating the items under analysis. The amounts harvested from any surface are very minute, yet sufficient for analysis via advanced mass spectrometry instrumentation, thus permitting the identification of all captured material. It is hoped that the present review will stimulate the scientific community to adopt it for projects pertaining to Cultural Heritage.

Il n'y a pas d'amour heureux pour Casanova: Chemical- and bio-analysis of his Memoirs.

The original manuscript of Casanova's Memoirs is stored at the Bibliothèque Nationale de France in Paris. We have gained access to it and explored the surfaces of chapters one and two (via the ethylene vinyl acetate [EVA] film technology, i.e., of diskettes of ethylene vinyl acetate with embedded strong cation and anion exchangers and C8 resins) in search of potential diseases of the author, especially of the gonorrhea bacterium, since Casanova reported that he had several bouts of this pathology along his adventurous life. Although the bacterium was not found, we have detected high levels of HgS as red spots along the lines of the manuscript, suggesting that Casanova was using this chemical as a cure for his venereal disease. Additionally, among the several bacteria identified on the surface via mass spectrometry, we could detect traces of Streptococcus uberis, a typical animal infection, found also in humans, together with a few strains of Lactobacilli, probably present in his saliva. The EVA film technology appears to open new horizons for investigating the world Cultural Heritage.

CASAnova: a multiclass support vector machine model for the classification of human sperm motility patterns.

The ability to accurately monitor alterations in sperm motility is paramount to understanding multiple genetic and biochemical perturbations impacting normal fertilization. Computer-aided sperm analysis (CASA) of human sperm typically reports motile percentage and kinematic parameters at the population level, and uses kinematic gating methods to identify subpopulations such as progressive or hyperactivated sperm. The goal of this study was to develop an automated method that classifies all patterns of human sperm motility during in vitro capacitation following the removal of seminal plasma. We visually classified CASA tracks of 2817 sperm from 18 individuals and used a support vector machine-based decision tree to compute four hyperplanes that separate five classes based on their kinematic parameters. We then developed a web-based program, CASAnova, which applies these equations sequentially to assign a single classification to each motile sperm. Vigorous sperm are classified as progressive, intermediate, or hyperactivated, and nonvigorous sperm as slow or weakly motile. This program correctly classifies sperm motility into one of five classes with an overall accuracy of 89.9%. Application of CASAnova to capacitating sperm populations showed a shift from predominantly linear patterns of motility at initial time points to more vigorous patterns, including hyperactivated motility, as capacitation proceeds. Both intermediate and hyperactivated motility patterns were largely eliminated when sperm were incubated in noncapacitating medium, demonstrating the sensitivity of this method. The five CASAnova classifications are distinctive and reflect kinetic parameters of washed human sperm, providing an accurate, quantitative, and high-throughput method for monitoring alterations in motility.

Light-Inducible CRISPR Labeling.

CRISPR labeling is a powerful technique to study the chromatin architecture in live cells. In CRISPR labeling, a catalytically dead CRISPR-Cas9 mutant is employed as programmable DNA-binding domain to recruit fluorescent proteins to selected genomic loci. The fluorescently labeled loci can then be identified as fluorescent spots and tracked over time by microscopy. A limitation of this approach is the lack of temporal control of the labeling process itself: Cas9 binds to the g(uide)RNA-complementary target loci as soon as it is expressed. The decoration of the genome with Cas9 molecules will, however, interfere with gene regulation and-possibly-affect the genome architecture itself. The ability to switch on and off Cas9 DNA binding in CRISPR labeling experiments would thus be important to enable more precise interrogations of the chromatin spatial organization and dynamics and could further be used to study Cas9 DNA binding kinetics directly in living human cells.Here, we describe a detailed protocol for light-inducible CRISPR labeling. Our method employs CASANOVA, an engineered, optogenetic anti-CRISPR protein, which efficiently traps the Streptococcus pyogenes (Spy)Cas9 in the dark, but permits Cas9 DNA targeting upon illumination with blue light. Using telomeres as exemplary target loci, we detail the experimental steps required for inducible CRISPR labeling with CASANOVA. We also provide instructions on how to analyze the resulting microscopy data in a fully automated fashion.